Method for treating cartilage disorders

ABSTRACT

The present invention relates to a method for the treatment of a cartilage disorder, including cartilage damaged by injury or degenerative cartilagenous disorders. The method involves contacting the cartilage with an IGF-1 analog with altered affinity for IGF-binding proteins (IGFBPs) or an IGFBP displacer peptide that prevents the interaction of an IGF with an IGFBP and does not bind to a human IGF receptor.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/858,935 filed May 16, 2001 which claims priority to provisionalapplication No. 60/248,985, filed Nov. 15, 2000 and No. 60/204,490,filed May 16, 2000, and is a continuation-in-part of U.S. applicationSer. No. 09/337,227, filed Jun. 22, 1999, which claims priority to U.S.application Ser. No. 08/825,852, filed Apr. 4, 1997 and Ser. No.09/052,888, filed Mar. 31, 1998, and a continuation-in-part of U.S.application Ser. No. 09/052,888, filed Mar. 31, 1998, which claimspriority to U.S. application Ser. No. 08/825,852, filed Apr. 4, 1997,and is a continuation-in-part of U.S. application Ser. No. 09/477,923,filed Jan. 5, 2000, which claims priority to provisional application No.60/115,010, filed Jan. 6, 1999, and is a continuation-in-part of U.S.application Ser. No. 09/477,924, filed Jan. 5, 2000, which claimspriority to provisional application No. 60/115,010, filed Jan. 6, 1999and No. 60/170,261, filed Dec. 9, 1999, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the treatment ofcartilage disorders, including stimulation of cartilage repair andtreatment of degenerative cartilagenous disorders.

BACKGROUND OF THE INVENTION

[0003] Degenerative cartilagenous disorders broadly describe acollection of diseases characterized by degeneration or metabolicabnormalities of the connective tissues that are manifested by pain,stiffness and limitation of motion of the affected body parts. Theorigin of these disorders can be pathological or as a result of traumaor injury.

[0004] Osteoarthritis (OA), also known as osteoarthrosis or degenerativejoint disease, is the result of a series of localized degenerativeprocesses that affect the articular structure and result in pain anddiminished function. The incidence of OA increases with age, andevidence of OA involvement can be detected in some joints in themajority of the population by age 65. OA is often also accompanied by alocal inflammatory component that may accelerate joint destruction.

[0005] OA is characterized by disruption of the smooth articulatingsurface of cartilage, followed by formation of clefts and fibrillation,and ultimately by the full-thickness loss of the cartilage. Coincidentwith the cartilaginous changes are alterations of the periarticularbone. These include the development of palpable bone enlargements at thejoint margins and deformity resulting from assymetric cartilagedestruction. OA symptoms include local pain at the affected joints,especially after use. With disease progression, symptoms may developinto a continuous aching sensation, local discomfort, and cosmeticalterations of the affected joint.

[0006] In contrast to the localized disorder OA, rheumatoid arthritis(RA) is a systematic destructive and debilitating disease that isbelieved to begin in the synovium, the tissues surrounding the joint.The prevalance of RA is about ⅙ that of OA in the general population ofthe United States. It is a chronic autoimmune disorder characterized bysymmetrical synovitis of the joint and typically affects small and largediarthrodial joints, leading to their progressive destruction. As thedisease progresses, the symptoms of RA may also include fever, weightloss, thinning of the skin, multi-organ involvement, scleritis, cornealulcers, the formation of subcutaneous or subperiosteal nodules, andpremature death.

[0007] The response of normal patients (e.g., preinjury or predisease)to injury or arthritic degeneration is often sub-optimal. Thebiochemical and mechanical properties of this damaged cartilage differfrom those of normal cartilage, resulting in inadequate or alteredfunction. This damaged cartilage, termed herein “fibrocartilage,” doesnot approximate the durability and function of normal cartilage.

[0008] Since cartilage is avascular and mature chondrocytes have littleintrinsic potential for replication, mature cartilage has limitedability for repair. Thus, damage to the cartilage layer that does notpenetrate to the subchondral bone does not undergo efficient repair. Incontrast, when the subchondral bone is penetrated, its vascular supplyallows a triphasic repair to take place. The resulting tissue is usuallymechanically sub-optimal fibrocartilage.

[0009] The degradation associated with osteoarthritis usually initiallyappears as fraying and fibrillation of the surface. Loss of proteoglycanfrom the matrix also occurs. As the surface fibrillation progresses, thedefects penetrate deeper into the cartilage, resulting in loss ofcartilage cells and matrix. The subchondral bone thickens, is slowlyexposed, and may appear polished. Bony nodules or osteophytes also oftenform at the periphery of the cartilage surface and occasionally growover the adjacent eroded areas. If the surface of these bony outgrowthsis permeated, vascular outgrowth may occur and cause the formation oftissue plugs containing fibrocartilage.

[0010] The transplantation of chondrocytes is known as a means ofstimulating cartilage repair. However, the possibility of the host'simmunogenic response as well as the possible transmission of viral andother infectious diseases makes this method less desirable. These riskscan be minimized to some extent with allograft and autogenoustransplants; however, the culturing and growth of patient-specific cellsis cost prohibitive on a mass scale.

[0011] Other methods of stimulating cartilage repair include theantagonism of molecules that are associated with or aggravate cartilagedestruction, for example, interleukin-1-alpha (IL-1α) and nitric oxide(NO). The cytokine IL-1α has catabolic effects on cartilage, includingthe generation of synovial inflammation and up-regulation of matrixmetalloproteinases and prostaglandin expression (Baragi et al., J. Clin.Invest., 96: 2454-2460 (1995); Baragi et al., Osteoarthritis Cartilage,5: 275-282 (1997); Evans et al., J. Leukoc. Biol., 64: 55-61 (1998);Evans and Robbins, J. Rheumatol., 24: 2061-2063 (1997); Kang et al.,Biochem. Soc. Trans., 25: 533-537 (1997); Kang et al., OsteoarthritisCartilage, 5: 139-143 (1997)). One means of antagonizing IL-1α isthrough application of soluble IL-1 receptor antagonist (IL-1ra), anaturally-occurring protein that inhibits the effects of IL-1 bypreventing IL-1 from binding to and activating its receptor onchondrocytes and synoviocytes, thereby lowering the effectiveconcentration of IL-1.

[0012] NO plays a substantial role in the destruction of cartilage (Aminet al., Curr. Opin. Rheum., 10: 263-268 (1998)). Cartilage obtained fromosteoarthritic joints endogenously produces large amounts of NO. Normalcartilage does not produce NO unless stimulated with cytokines such asIL-1, while osteoarthritic cartilage explants continue to express NOsynthase for up to 3 days in culture despite the absence of addedstimuli. Moreover, the inhibition of NO has been shown to preventIL-1α-mediated cartilage destruction and chondrocyte death as well asthe progression of osteoarthritis.

[0013] The ability of peptide growth factors to promote repair ofdamaged cartilage has also been examined. Peptide growth factors arevery significant regulators of cartilage growth and cell behavior (i.e.,differentiation, migration, division, or matrix synthesis and/orbreakdown) (Chen et al., Am J. Orthop., 26: 396-406 (1997)). Thesefactors are under investigation for their potential to induce hostcartilage repair without transplantation of cells, and are beingincorporated into engineered devices for implantation.

[0014] Because growth factors are soluble proteins of relatively smallmolecular mass that are rapidly absorbed and/or degraded, a greatchallenge exists in making them available to cells in sufficientquantity and for sufficient duration. It is likely desirable to havedifferent factors present at the repair site during different parts ofthe developmental cycle, and for varying lengths of time. The idealdelivery vehicle is biocompatible and resorbable, has the appropriatemechanical properties, and results in no harmful degradation products.Growth factors that previously have been proposed to stimulate cartilagerepair include insulin-like growth factor-I (IGF-1) (Osborn, J. Orthop.Res., 7: 35-42 (1989); Florini and Roberts, J. Gerontol., 35: 23-30(1980); U.S. Pat. No. 5,843,899), basic fibroblast growth factor (bFGF),[Toolan et al., J. Biomec. Mat. Res., 4]: 244-50 (1998); Sah et al.,Arch. Biochem. Biophys., 308: 137-47 (1994)), bone morphogenetic protein(BMP) (Sato and Urist, Clin. Orthop. Relat. Res., 183: 180-187 (1984);Chin et al., Arthritis Rheum. 34: 314-324 (1991)), and transforminggrowth factor beta (TGF-β) (Hill and Logan, Prog. Growth Fac. Res., 4:45-68 (1992); Guerne et al., J. Cell Physiol., 158: 476-484 (1994); Vander Kraan et al., Ann. Rheum. Dis., 51: 643-647 (1992)).

[0015] It has been well established that the GH/IGF/IGFBP system isinvolved in the regulation of anabolic and metabolic homeostasis andthat defects in this system may adversely affect growth, physiology, andglycemic control (Jones et al., Endocr. Rev., 16: 3-34 (1995); Davidson,Endocr. Rev., 8: 115-131 (1987); Moses, Curr. Opin. Endo. Diab., 4:16-25 (1997)). It has been proposed that IGF-1 could be useful for thetreatment or prevention of osteoarthritis, because of its ability tostimulate both matrix synthesis and cell proliferation in culture(Osborn, J. Orthop. Res., 7: 35-42 (1989)). IGF-1 has been administeredwith sodium pentosan polysulfate (PPS) (a chondrocyte catabolic activityinhibitor) to severely osteoarthritic canines with the effect ofreducing the severity of the disease perhaps by lowering the levels ofactive neutral metalloproteinase in the cartilage. In the model ofmildly osteoarthritic canines, therapeutic intervention with IGF-1 andPPS together appeared to successfully maintain cartilage structure andbiochemistry, while IGF alone was ineffective, as described inRogachefsky, Osteoarthritis and Cartilage, 1: 105-114 (1993);Rogachefsky et al., Ann. NY Acad. Sci., 732: 392-394 (1994). The use ofIGF-1 either alone or as an adjuvant with other growth factors tostimulate cartilage regeneration has been described in WO 91/19510, WO92/13565, U.S. Pat. No. 5,444,047, and EP 434,652.

[0016] IGF-1 has also been found useful in the treatment of osteoporosisin mammals exhibiting decreased bone mineral density and those exposedto drugs or environmental conditions that result in bone densityreduction and potentially osteoporosis, as described in EP 560,723 andEP 436,469.

[0017] IGF-1 insufficiency may have an etiologic role in the developmentof osteoarthritis (Coutts et al., “Effect of growth factors on cartilagerepair,” Instructional Course Lect., 47: 487-494 (Amer. Acad. Orthop.Surg.: Rosemont, Ill. 1997)). Some studies indicate that serum IGF-1concentrations are lower in osteoarthritc patients than control groups,while other studies have found no difference. Nevertheless, it has beenshown that both serum IGF-1 levels and chrondrocyte responsiveness toIGF-1 decrease with age, with the latter likely due to high levels ofIGF binding proteins (IGFBPs) (Florini and Roberts, J. Gerontol., 35:23-30 (1980); Martin et al., J. Orthop. Res., 15: 491-498 (1997);Fernihough et al., Arthr. Rheum. 39: 1556-1565 (1996)). Thus, both thedecreased availability of IGF-1 as well as diminished chondrocyteresponsiveness/disregulation of IGFBPs thereto may contribute to theimpaired cartilage matrix homeostasis and tissue degeneration thatoccurs with advancing age and disease.

[0018] Of the IGFBPs, IGFBP-3 appears to be the most responsible forregulating the total levels of IGF-1 and IGF-2 in plasma. IGFBP-3 is aGH-dependent protein and is reduced in cases of GH-deficiency orresistance (Jones et al., supra; Rosenfield et al., “IGF-1 treatment ofsyndromes of growth hormone insensitivity” In: The insulin-like growthfactors and their regulatory proteins, Eds Baxter R C, Gluckman P D,Rosenfield R G. Excerpta Medica, Amsterdam, 1994), pp 457-464; Scharf etal., J. Hepatology, 25: 689-699 (1996)). IGFBPs are able to enhance orinhibit IGF activity, depending largely on their post-translationalmodifications and tissue localization (reviewed in Jones and Clemmons,Endocr. Rev. 16:3-34 (1995); Collett-Solberg and Cohen, Endocrinol.Metabol. Clin. North Am. 25:591-614 (1996)). In addition, disregulationin IGFBPs (-3, -4 and/or -5) may play a key role in arthritic disorders(Chevalier and Tyler, Brit. J. Rheum. 35: 515-522 (1996); Olney et al.,J. Clin. Endocrinol. Metab. 81: 1096-1103 (1996); Martel-Pelletier etal., Inflamm. Res., 47: 90-100 (1998)). It has been reported that IGF-1analogs with very low binding affinity for IGFBPs were more effectivethan wild-type IGF-1 in stimulating proteoglycan synthesis (Morales,Arch Biochem. Biophys. 343(2), 164-172 (1997)). More recent data,however, suggest that IGFBPs contribute to IGF binding to and transportthrough cartilage tissue, and IGFBPs may thus regulate bioavailabilityof IGF-1 within the joint (Bhakta et al., J. Biol. Chem., 275: 5860-5866(2000)).

[0019] The biodistribution of IGF-1 critically depends on (a) theformation of long-lived high molecular weight complexes and (b) theabsolute IGFBP concentrations. The majority of IGF-1 in the circulationis found in complex with IGFBP-3 and a third protein termed acid-labilesubunit (ALS) (Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995);Clemmons, Cytokine Growth Factor Rev., 8: 45-62 (1997); Jones andClemmons, Endocr. Rev., 16: 3-34 (1995)). This ternary complex of 150-kDmolecular weight is unable to traverse the vasculature walls and acts asa circulating reservoir for IGF's. As a consequence, the serum half-lifeof IGF-1 in ternary complexes is reported to be 12-15 hours, as opposedto 30 minutes in binary complexes, or 10 minutes in the free form(Simpson et al., Growth Horm IGF Res, 8: 83-95 (1998); Twigg and Baxter,J. Biol. Chem., 273: 6074-6079 (1998)).

[0020] IGFBP-3 and -5 are apparently unique in their ability to form aternary complex with ALS. ALS association occurs only in the presence ofIGF-1, and a basic motif in the carboxy-terminal domains of IGFBP-3 and-5 seems to mediate this interaction (Baxter et al., J. Biol. Chem.,267: 60-65 (1992); Firth et al., J. Biol. Chem., 273: 2631-2638 (1998);Twigg and Baxter, supra).

[0021] The second determinant of IGF-1 biodistribution is the totalconcentration of binding proteins: IGFBP-3 is the most abundant bindingprotein, followed by IGFBP-1 and -2 levels, whereas the serumconcentrations of IGFBP-4, -5, and 6 are quite low (Clemmons, CytokineGrowth Factor Rev., 8: 45-62 (1997)). IGFBP-3 therefore represents themain IGF-1 carrier in the blood. In contrast, a substantial portion ofIGFBP-1 and -2 in the blood are unoccupied. Hence, they appear to be themajor modulators of free IGF-1 levels (Clemmons, 1997, supra).

[0022] WO 94/04569 discloses a specific binding molecule, other than anatural IGFBP, that is capable of binding to IGF-1 and can enhance thebiological activity of IGF-1. WO 98/45427 published Oct. 15, 1998;Lowman et al., Biochemistry, 37: 8870-8878 (1998); and Dubaquié andLowman, Biochemistry, 38: 6386 (1999) disclose IGF-1 agonists identifiedby phage display. Also, WO 97/39032 discloses ligand inhibitors ofIGFBP's and methods for their use. Further, U.S. Pat. No. 5,891,722discloses antibodies having binding affinity for free IGFBP-1 anddevices and methods for detecting free IGFBP-1 and a rupture in a fetalmembrane based on the presence of amniotic fluid in a vaginal secretion,as indicated by the presence of free IGFBP-1 in the vaginal secretion.WO 00/23469 published Apr. 27, 2000 discloses fragments of IGFBPs andanalogs of IGF-1 for use in, e.g., cancer, ischemic injury, and diabetestreatment.

[0023] There exists a continuing need for an effective therapy for thetreatment and repair of cartilage, including cartilage damaged as aresult of injury and/or disease.

SUMMARY OF THE INVENTION

[0024] Accordingly, the present invention concerns a method of treatinga cartilage disorder as claimed, comprising contacting cartilage with aneffective amount of an active agent selected from an IGF-1 analog with abinding affinity preference for IGFBP-3 over IGFBP-1, an IGF-1 analogwith a binding affinity preference for IGFBP-1 over IGFBP-3, or an IGFBPdisplacer peptide that prevents the interaction of IGF with IGFBP-3 orIGFBP-1 and does not bind to a human IGF receptor. Preferably, thecartilage is treated in vivo in a mammal and the active agent isadministered to the mammal. Also, the active agent is optionallycontacted with the cartilage in an extended-release form and/oradministered locally to the joint alone or, if the active agent is anIGFBP displacer peptide or IGF-1 analog with a preference for IGFBP-3over IGFBP-1, together with IGF-1 and/or ALS, preferably human,native-sequence IGF-1 if the mammal is human.

[0025] Preferably, the active agent is an IGF-1 variant wherein theamino acid residue at position 3, 7, 10, 16, 25, or 49, or the aminoacid residues at positions 3 and 49 of native-sequence human IGF-1 arereplaced with an alanine, a glycine, or a serine residue, or an IGF-1variant wherein the amino acid residue at position 9, 12, 15, or 20 isreplaced with a lysine or arginine residue, or an IGFBP-3 displacerpeptide designated as: Y24LY31A IGF-1; 4D3.3P; BP3-4D3.11;BP3-4D3.11DEL; BP3-4B3.3; BP3-01-ox; BP3-02-ox; BP3-06; BP3-08; BP3-15;BP3-16; BP3-17; BP3-25; BP3-27; BP3-28; BP3-30; BP3-39; BP3-40; BP3-41;BP3-107; or BP3-108; or an IGFBP-1 displacer peptide designated as:BP1-01; BP1-02; BP1-04; BP1-10; BP1-11; BP1-12; BP1-13; BP1-14; BP1-15;BP1-16; BP1-17; BP1-18; BP1-19; BP1-20; BP1-21A; BP1-21B; BP1-25;BP1-40; BP67; BP68; BP1-625; BP1-625-Z; BP1-625T; BP1027; BP1028;BP1029; BP1030; (i+7)D; (i+8)B; and (i+8)C.

[0026] The letter followed by a number followed by a letter indicates anIGF-1 analog wherein the amino acid letter to the left of the number isthe original amino acid in native-sequence human IGF-1, the number isthe position where the amino acid is changed, and the amino acid letterto the right of the number is the substituted amino acid. Hence, forexample, F49A indicates an IGF-1 variant wherein the phenylalanineresidue at position 49 of native-sequence human IGF-1 is changed to analanine residue, and E3AF49A indicates an IGF-1 variant wherein theglutamine residue at position 3 of native-sequence human IGF-1 ischanged to an alanine residue, and the phenylalanine residue at position49 of native-sequence human IGF-1 is changed to an alanine residue.

[0027] In another embodiment, the above method is for the treatment ofcartilage damaged or diseased as a result of a degenerativecartilagenous disorder. Preferably, the disorder is an articularcartilage disorder, and most preferably is OA or RA.

[0028] In a further embodiment, the above method is for the treatment ofjoints damaged directly or indirectly by injury, preferably microdamageor blunt trauma, a chondral fracture, an osteochondral fracture.

[0029] Optionally, the invention concerns the above treatment methodwherein the cartilage is contacted with an effective amount of the IGF-1analog or IGFBP displacer peptide as defined above in combination withan effective amount of a cartilage growth factor or cartilage catabolismantagonist.

[0030] In another embodiment, the invention concerns a method ofmaintaining, enhancing, or promoting the growth of chondrocytes inserum-free culture by contacting the chondrocytes with an effectiveamount of an IGF-1 analog or an IGFBP displacer peptide as identifiedabove. Alternatively, the method concerns contacting the chondrocytewith an effective amount of an IGF-1 analog or an IGFBP displacerpeptide in an extended-release formulation. Alternatively, the presentinvention concerns a method of stimulating the regeneration orpreventing the degradation of cartilage resulting from injury or adegenerative cartilagenous disorder by transplantation of an effectiveamount of chondrocytes previously treated with an effective amount of anIGF-1 analog or an IGFBP displacer peptide as defined above.

[0031] In another embodiment, the present invention concerns an articleof manufacture comprising a container holding an IGF-1 analog or anIGFBP displacer peptide as defined above in a pharmaceuticallyacceptable carrier with instructions for its use in treating a cartilagedisorder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A-1C depict the DNA sequence (SEQ ID NO:1) of plasmidpt4.g8 used as a template to construct a phage library. Also shown isthe amino acid sequence (SEQ ID NO:2) of an antibody-recognizable(gD-tag) peptide fused to g8p of bacteriophage M13.

[0033]FIG. 2 shows gene-8 naive phage library enrichments with aselection using four library pools each and the targets IGF-1, IGFBP-1,and IGFBP-3.

[0034]FIG. 3 shows an IGF-1 blocking assay using g8-phage peptides fromIGFBP-3 selections, where the phage titration is with 100 nM IGF-1. Inthe Figure, the open circles are peptide 4A3.1, the open triangles arepeptide 4B3.4, the open squares are peptide 4C3.2, the solid circles arepeptide 4D3.3, the solid triangles are peptide 4D3.4, and the solidsquares are peptide 4D3.5.

[0035]FIG. 4 shows an IGF-1 blocking assay using g8-phage peptides fromIGFBP-3 selections, where the phage titration is without IGF-1. Thedesignations for the peptides are the same as those described above forFIG. 3.

[0036]FIG. 5 shows an IGF-1 blocking assay using g8-phage peptides fromIGFBP-3 selections, where the peptides (4C3.2, 4D3.8, 4D3.9, 4D3.11, and4D3.12) are from a NEUTRAVIDIN™/DTT selection. The solid bars are with100 μM IGF-1 and the open bars are without IGF-1.

[0037]FIG. 6 shows an IGF-1 blocking assay using g8-phage peptides fromIGFBP-3 selections where the peptides (indicated on the x axis) are fromdirect-coat/HCl selection. The solid bars are with 100 μM IGF-1 and theopen bars are without IGF-1.

[0038]FIG. 7 depicts a competition assay of IGFBP-3 inhibition by apeptide binding to IGFBP-3 (designated BP3-01) using a BIACORE™surface-plasmon-resonance device to measure free binding protein. Thecircles indicate 800 response units (RU) of IGF-1 and the squaresindicate 400 RU of immobilized IGF-1.

[0039]FIG. 8 depicts a competition assay of IGFBP-3 inhibition by apeptide binding to IGFBP-3 (designated BP3-02) using a BIACORE™surface-plasmon-resonance device to measure free binding protein. Thecircles indicate 800 RU of IGF-1 and the squares indicate 400 RU ofimmobilized IGF-1.

[0040]FIG. 9 shows a radiolabeled IGF-1 plate assay of the ability oftwo peptides that bind to IGFBP-3 but not to the Type 1 IGF receptor(BP3-01-ox: circles, and BP3-02-ox: squares) to inhibit IGFBP-3.

[0041]FIG. 10 shows a radiolabeled IGF-1 plate assay of the ability ofthe two IGFBP-3 binding peptides described for FIG. 9 to inhibit IGFBP-1(symbols are the same).

[0042] FIGS. 11A-11D depict KIRA assays of IGF-1 activity using threepeptides (BP1-01: squares, BP1-02: circles, and BP03-ox: triangles).FIG. 11A depicts the peptides alone, FIG. 11B depicts the peptides plusIGF-1 plus IGFBP-1, FIG. 11C depicts the peptides plus IGF-1, and FIG.11D depicts the peptides plus IGF-1 plus IGFBP-3.

[0043]FIG. 12 depicts an IGF-2 competition assay of IGFBP-3 inhibitionby four peptides, designated BP3-01-ox (open squares), BP3-14 (opencircles), BP3-15 (closed circles), and BP3-17 (closed squares), using aBIACORE™ surface-plasmon-resonance device to measure free bindingprotein. Each peptide was tested using 20 nM IGFBP-3 and approximately1500 RU of immobilized IGF-2.

[0044]FIGS. 13A and 13B show a phage ELISA of the variant, G15-A70VIGF-1, binding to IGFBP-1 (FIG. 13A) and IGFBP-3 (FIG. 13B). Microtiterplates coated with 1 μg/ml IGFBP-1 (FIG. 13A) or IGFBP-3 (FIG. 13B) wereincubated with phage particles displaying G15-A70V in the presence ofthe indicated amounts of soluble competitor protein, IGFBP-1 (FIG. 13A)or IGFBP-3 (FIG. 13B). The half-maximal inhibitory concentration (IC₅₀)of competitor, i.e., the inhibitory concentration of competitor thatresulted in half-maximal binding of the phagemid in that particularexperiment, is denoted for the respective IGFBP.

[0045]FIG. 14 shows the loss or gain of IGFBP affinity for the IGF-1mutants tested by phage ELISA. Relative IC₅₀ values(IC_(50mut)/IC_(50 G15-A70V)) of each IGF-1 alanine mutant (affinitychanges of each mutant for the binding proteins with respect to IGF-1G15-A70V) are shown for IGFBP-1 (filled bars) and IGFBP-3 (open bars).Data are taken from Table I below. Relative IC₅₀ values <1 denote gainof affinity; values >1 denote loss of affinity. The asterisk indicatesthat these particular variants were not displayed on phage, as judged byantibody binding.

[0046]FIGS. 15A and 15B show binding specificity of the IGF-1 variantF49A displayed on phage to IGFBP-1 and -3, respectively, incompetitive-phage ELISA. Phagemid particles displaying F49A (squares)were bound to plates coated with IGFBP-3 in the presence of theindicated amounts of soluble IGFBP-1 (FIG. 15A) or IGFBP-3 (FIG. 15B).The same experiment was carried out in parallel with phage displayingthe wild-type-like IGF-1 variant G15-A70V (circles). See Tables I and IIbelow for absolute IC₅₀ values. Data points are mean±standard deviation,n=2. Immunosorbent plates were coated with 1 μg/ml IGFBP-3 and ELISAwere carried out as described in the Examples below using wild-typeIGF-1 phage (WT, circles) and IGF-F49A phage (F49A, squares) inparallel. Experiments were carried out in duplicate, and data points areshown as mean±standard deviation.

[0047]FIG. 16 discloses a sequence alignment of native-sequence humanIGF-1 (designated wtIGF)(SEQ ID NO:3), native-sequence human proinsulin(designated proinsulin) (SEQ ID NO:4), and native-sequence human insulin(designated insulin (B chain) followed by insulin (A chain)) (SEQ IDNO:5). The asterisks and dots indicate sequence identity and sequencesimilarity, respectively, at the indicated amino acid positions amongthe three sequences.

[0048] FIGS. 17A-17D show a biosensor analysis of IGFBP binding toimmobilized IGF-1 variants. Sensorgrams are shown for IGFBP-1 (FIGS.17A, 17C) or IGFBP-3 (FIGS. 17B, 17D) binding to immobilized wild-typeIGF-1 (FIGS. 17A, 17B) or F49A IGF variant (FIGS. 17C, 17D). Theconcentrations of ligand in each experiment were 1 μM, 500 nM, and 250nM. See Table II for kinetic parameters.

[0049] FIGS. 18A-18B show a model of the functional binding epitopes forIGFBP-1 and IGFBP-3, respectively, on the surface of IGF-1. Amino acidside chains were classified according to their relative contribution inbinding energy (Table I) and colored as follows: no effect (grey); 2-5fold loss of apparent affinity (yellow); 5-10 fold (orange); 10-100 fold(bright red); >100 fold (dark red). If available, numbers from phageELISA experiments in Table I below were used. BIACORE™ data were usedinstead for V11A, R36A, and P39A variants (Table II). The NMR structureof IGF-1 (Cooke et al., Biochemistry, 30: 5484, (1991)) was representedusing the program Insight II™ (MSI, San Diego, Calif.). The bindingepitope for IGFBP-1 (FIG. 18A) is located on the “upper” and “lower”face of the N-terminal helix (residues 8-17), connected by theenergetically-important residue F49. For IGFBP-3 (FIG. 18B), individualIGF-1 side chains contribute very little binding energy. The bindingepitope has shifted away from the N-terminus and newly includes G22,F23, Y24.

[0050]FIG. 19 shows the amount of bound IGFBP-1, determined in acompetitive BIACORE™ binding experiment, plotted against the IGF variantconcentration for E3A/F49A (squares) and F49A (circles).

[0051]FIGS. 20A and 20B show, respectively, the calculated IGF-1activity in nM units for several IGF-1 variants at 13 nM (high) and 1.3nM (low) variant concentrations using IGF-1 KIRA optical densityanalysis. The signal obtained for each IGF variant was compared to thatof a standard-dilution series of wild-type IGF-1, and reported in termsof an apparent IGF-1 concentration corresponding to the observedactivity.

[0052]FIGS. 21A and 21B show IGF receptor activation curves for F49AIGF-1 (FIG. 21A) and E3A/F49A (FIG. 21B) as well as for wild-type IGF-1,as measured using serial dilutions in KIRA assays. The variants arerepresented by squares and the wild-type IGF-1 is represented bycircles.

[0053]FIGS. 22A and 22B show an assessment of preliminarypharmacological properties of F49A and E3A/F49A IGF-1, radiolabeled andadministered intravenously to rats. FIG. 22A shows a time course of therate at which both molecules are cleared from the blood of the animals,where the squares represent wild-type IGF-1, the circles representE3A/F49A IGF-1, and the diamonds represent F49A IGF-1. FIG. 22B showsthe tissue-to-blood ratio for these two IGF variants in differentorgans, namely, kidney, liver, spleen, heart, and pancreas, at 5, 15,and 30 minutes, where the solid bars represent wild-type IGF-1, thedotted bars represent E3A/F49A IGF-1, and the striped bars representF49A IGF-1.

[0054]FIG. 23 shows circular dichroism spectra of wild-type IGF-1(circles), F49A IGF-1 (squares), and E3A/F49A IGF-1 (diamonds).

[0055]FIG. 24 is a bar graph showing the effect of control, wild-typeIGF-1, F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) oncartilage matrix breakdown (proteoglycan release at 72 hours).

[0056]FIG. 25 is a bar graph showing the effect of wild-type IGF-1,F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) onIL1α-induced cartilage breakdown at 72 hours.

[0057]FIG. 26 is a bar graph showing the effect of control, wild-typeIGF-1, F49A, E3A/F49A (at a concentration of 40 or 400 ng/ml) on matrixsynthesis.

[0058]FIG. 27 is a bar graph showing the effect of wild-type IGF-1,F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) onIL1α-induced inhibition of matrix synthesis.

[0059]FIG. 28 is a bar graph showing the effect of control, wild-typeIGF-1, F49A and E3A/F49A (at a concentration of 40 or 400 ng/ml) onnitric oxide release.

[0060]FIG. 29 is a bar graph showing the effect of wild-type IGF-1,F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) onIL1α-induced nitric oxide production.

[0061]FIGS. 30A and 30B show the binding curves for phage particlesdisplaying either wild-type IGF-1 (circles), D12K (squares), or D12R(diamonds) bound to immobilized IGFBP-1 (FIG. 30A) or IGFBP-3 (FIG.30B).

[0062] FIGS. 31A-31D show the effects on porcine articular cartilageexplants cultured in media (−) or media with D12K, D12R, or wild-typeIGF-1 (at 10 nM) alone (FIGS. 31A, 31C) or in the presence of IL-1α (+a)at 1 ng/ml (FIGS. 31B, 31D).

[0063]FIG. 32 shows the effect on articular cartilage matrix synthesisin human tissue from diseased joints cultured in media alone (−) or withF49, E3A/F49, F16/F49, D12K, D12R or wild-type IGF-1 (at 40 ng/ml).

[0064] FIGS. 33A-33D show the effect on human articular cartilageexplants cultured in media (−) or treated with wild-type IGF-1 by itselfor in combination with either BP3-40 (FIGS. 33A, 33B) or BP3-15 (FIGS.33C, 33D) (at 0.1 mg/ml).

[0065] FIGS. 34A-34C show the trimeric complex formation of F49A orE3A/F49A with IGFBP-3 and ALS. IGFBP-3 immobilized on a biosensor chipwas saturated by including 1 μM wild-type IGF-1 (FIG. 34A), F49A (FIG.34B), or E3A/F49A (FIG. 34C) in the running buffer. ALS was injected at98 nM, 148 nM, and 33 nM, monitoring real-time association anddissociation to the preformed binary complex.

[0066]FIG. 35 shows a BIAcore™ inhibition assay of IGF-1 activity usingseven different peptides (BP1-16: filled circles, (i+7)A: open circles,(i+7)B: open diamonds, (i+7)C: open triangles, (i+7)D: open squares,(i+8)B: filled squares, (i+8)C: filled triangles).

[0067]FIG. 36 shows a KIRA assay of peptide activity using fourdifferent peptides (BP1-16: circles, BP1-02: squares, BP1-25: triangles,and BP1-40: diamonds).

[0068]FIG. 37 shows an analytical HPLC run of the trypsin-cleavedBP1-625-Z fusion. The major peaks were identified by mass spectrometryas (A) Z-domain fragment and (B) BP1-625 peptide.

[0069]FIG. 38 shows a BIAcore™ inhibition assay of IGF-1 activity usingfour different peptides (BP1-01: circles, BP1-625: squares, BP1-21A:triangles, and BP1-25: diamonds).

[0070]FIG. 39 shows the effect on proteoglycan synthesis of articularcartilage explants from human joints removed from patients undergoingjoint replacement cultured with IGF-1 alone (IGF) at 40 ng/ml, or IGF-1with BP1-17, BP3-15, or BP1-16 (0.1 mg/ml), or IGF-1 with buffer(HEPES).

DETAILED DESCRIPTION OF THE INVENTION

[0071] I. Definitions

[0072] “IGF-1 analogs” are amino acid variants of native-sequence IGF-1,preferably variants of human wild-type IGF-1. The dissociation constant(K_(D)) Of wild-type IGF-1 was determined to be 13 nM for IGFBP-1 and1.5 nM for IGFBP-3. The difference in affinity for the IGFBP's is due toa 10-fold faster association rate (k_(a)) of IGF-1 to IGFBP-3 (3.2×10⁵versus 3.2×10⁴ M⁻¹s⁻¹). Such analogs may have one or more amino acidalterations as compared to native IGF-1. As used herein, the term “IGF-1analogs” refers either to an IGF-1 analog with a binding affinitypreference for IGFBP-3 over IGFBP-1 or an IGF-1 analog with a bindingaffinity preference for IGFBP-1 over IGFBP-3, as defined below.

[0073] An “IGF-1 analog with a binding affinity preference for IGFBP-3over IGFBP-1” refers to an IGF-1 analog that exhibits altered bindingaffinity for any one or more of the IGFBPs over that of native-sequenceIGF-1, such that the analog's relative binding affinity (R(3)) forIGFBP-3 [defined as R(3)=K_(D)(IGF-1:IGFBP-3)/K_(D)(analog:IGFBP-3)] isat least about 10-fold greater than its relative binding affinity (R(1))for IGFBP-1 [defined asR(1)=K_(D)(IGF-1:IGFBP-1)/K_(D)(analog:IGFBP-1)], as shown by, forexample, by kinetic analysis using a BIACORE™ instrument of theexpressed and purified analogs.

[0074] Conversely, an “IGF-1 analog with a binding affinity preferencefor IGFBP-1 over IGFBP-3” refers to an IGF-1 analog that exhibitsaltered binding affinity for any one or more of the IGFBPs over that ofnative-sequence IGF-1, such that the analog's relative binding affinity(R(1)) for IGFBP-1 [defined asR(1)=K_(D)(IGF-1:IGFBP-1)/K_(D)(analog:IGFBP-1)] is at least about10-fold greater than its relative binding affinity (R(3)) for IGFBP-3[defined as R(3)=K_(D)(IGF-1:IGFBP-3)/K_(D)(analog:IGFBP-3)], as shownby, for example, by kinetic analysis using a BIACORE™ instrument of theexpressed and purified analogs.

[0075] “Peptides” have at least two amino acids and include polypeptideshaving at least about 50 amino acids. The definition includes peptidederivatives, their salts, or optical isomers.

[0076] An IGFBP displacer peptide that “inhibits” or “prevents” theinteraction of an IGF with an IGFBP refers to a peptide that increasesserum and tissue levels of biologically active IGF, no matter how thisincrease occurs. For instance, the peptide may partially or completelydisplace active IGF from a complex in which the IGF is bound to anIGFBP. The peptide under this definition may bind to an IGFBP, andpossibly thereby act to displace an endogenous IGF formerly bound to theIGFBP. Alternatively, it may bind to IGF itself at a site remote fromthat involved in receptor interactions so as to inhibit or prevent theinteraction of the IGF with IGFBP, but not inhibit or prevent theinteraction of the IGF with any of its receptors. Further, while thepeptide will occupy the IGFBP-3 binding site, the effect on the ternarycomplex with ALS will depend on whether the binary complexes can formternary ones. Peptides that can form complexes with the ALS of theternary complex will replace IGFs but not affect the concentration ofIGFBP-3 or of ternary complexes. Peptides that cannot form complexeswith ALS will occupy IGFBP-3, and the amount of ALS/IGFBP-3/IGF complexwill be reduced. Preferably, the IGFBP displacer peptide is an IGFBP-3or IGFBP-1 displacer peptide.

[0077] A peptide that “binds to IGFBP-3” or “binds to IGFBP-l” refers toa peptide that binds IGFBP-3 or IGFBP-1 to at least some degree, whetherwith high affinity or not.

[0078] As used herein, “human IGF receptor” refers to any receptor foran IGF found in humans and includes the Type 1 and Type 2 IGF receptorsin humans to which both human IGF-1 and IGF-2 bind, such as theplacental Type 1 IGF-1 receptor, etc.

[0079] A peptide that “does not bind to a human IGF receptor” does notbind at all to any such receptor, or binds to such receptor with anaffinity more than about 200-fold less than wild-type human IGF-1(hIGF-1) or wild-type human IGF-2 (hIGF-2) binds to such receptor.Preferably, the peptide binds to such receptor with an affinity of morethan about 250-fold less than wild-type hIGF-1 or hIGF-2 binds to thesame receptor or does not bind at all.

[0080] The term “cartilage disorder” refers to any injury or damage tocartilage, and to a collection of diseases that are manifested bysymptoms of pain, stiffness, and/or limitation of motion of the affectedbody parts. Included within the scope of “cartilage disorders” is“degenerative cartilagenous disorders”, which is a colllection ofdisorders characterized, at least in part, by degeneration or metabolicderangement of connective tissues of the body, including not only thejoints or related structures, including muscles, bursae (synovialmembrane), tendons, and fibrous tissue, but also the growth plate,meniscal system, and intervertebral discs.

[0081] In one embodiment, the term “degenerative cartilagenousdisorders” includes “articular cartilage disorders,” which arecharacterized by disruption of the smooth articular cartilage surfaceand degradation of the cartilage matrix. Additional pathologies includenitric oxide production, and inhibition or reduction of matrixsynthesis. Included within the scope of “articular cartilage disorder”are OA and RA. Examples of degenerative cartilagenous disorders includesystemic lupus erythematosus and gout, amyloidosis or Felty's syndrome.Additionally, the term covers the cartilage degradation and destructionassociated with psoriatic arthritis, kidney disorders, osteoarthrosis,acute inflammation (e.g., yersinia arthritis, pyrophosphate arthritis,gout arthritis (arthritis urica), and septic arthritis), arthritisassociated with trauma, ulcerative colitis (e.g., Crohn's disease),multiple sclerosis, diabetes (e.g., insulin-dependent and non-insulindependent), obesity, giant cell arthritis, and Sjögren's syndrome. Inone preferred embodiment, the disorder is microdamage or blunt trauma, achondral fracture, or an osteochondral fracture.

[0082] “Osteoarthritis” or “OA” defines not a single disorder, but thefinal common pathway of joint destruction resulting from multipleprocesses. OA is characterized by localized assymetric destruction ofthe cartilage commensurate with palpable bone enlargements at the jointmargins. OA typically affects the interphalangeal joints of the hands,the first carpometacarpal joint, the hips, the knees, the spine, andsome joints in the midfoot, while large joints, such as the ankles,elbows, and shoulders, tend to be spared. OA can be associated withmetabolic diseases such as hemochromatosis and alkaptonuria,developmental abnormalities such as developmental dysplasia of the hips(congenital dislocation of the hips), limb-length descrepancies,including trauma and inflammatory arthritides such as gout, septicarthritis, and neuropathic arthritis. OA may also develop after extendedmechanical instability, such as resulting from sports injury or obesity.

[0083] “Rheumatoid arthritis” or “RA” is a systemic, chronic, autoimmunedisorder characterized by symmetrical synovitis of the joint andtypically affects small and large diarthroid joints alike. As RAprogresses, symptoms may include fever, weight loss, thinning of theskin, multiorgan involvement, scleritis, corneal ulcers, the formationof subcutaneous or subperiosteal nodules, and even premature death. Thesymptoms of RA often appear during youth and can include vasculitis,atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy,splenomegaly, leukopaenia, and chronic anaemia.

[0084] “Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathological condition or disorder.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. In treatment ofa degenerative cartilagenous disorder, a therapeutic agent may directlydecrease or increase the magnitude of response of a pathologicalcomponent of the disorder, or render the disease more susceptible totreatment by other therapeutic agents, e.g., antibiotics, antifungals,anti-inflammatory agents, chemotherapeutics, etc. The term “treatment”includes a method for the prevention of initial or continued damage ordisease of joints by degenerative cartilagenous disorders and/or injury.

[0085] The term “effective amount” is the minimum efficaciousconcentration of the IGF analog or IGFBP displacer peptide as set forthherein. This includes the minimum concentration of such protein orpeptide that causes, induces, or results in either a detectableimprovement or repair of damaged cartilage or a measurable protectionfrom continued or induced cartilage destruction, such as the inhibitionof synthesis or loss of proteoglycans from cartilage tissue.

[0086] “Cartilage growth factor” as used herein refers to agent(s) otherthan an IGF-1 analog or an IGFBP displacer peptide as identified hereinthat cause, induce, or result in an improvement in the condition of orprotection from initial or continued destruction of cartilage subject todamage by either injury or a degenerative cartilagenous disorder. Suchcartilage growth factors include insulin-like growth factors (e.g.,IGF-1, IGF-2), platelet-derived growth factors (PDGFs), bone morphogenicproteins (BMPs), transforming growth factor-βs (1-3), members of theepidermal growth factor family (e.g., EGF, HB-EGF, TGF-α), andfibroblast growth factors (FGFs).

[0087] “Cartilage catabolism antagonists” are those agents that inhibit,attenuate or otherwise block the activity or effect of molecules thatare associated with or aggravate cartilage destruction. For example,IL-1α and nitric oxide (NO) are agents known to be associated withcartilage destruction. Thus, direct (IL1ra) or indirect (IL-4 or IL-10)inhibitors of IL-1α or other inflammatory cytokines (e.g., TNF-α) and NOproduction would be considered “cartilage catabolism antagonists.”Moreover, antagonists of chondrocyte catabolism (e.g., sodium pentosanpolysulfate, glucosamine (and variants thereof, such as mannosamine) orchondroitin sulfate, tetracycline, hyaluronan) would also be consideredcartilage catabolism antagonists. Also included are agents that inhibitcatabolism of cartilage indirectly, for example through their effects onthe underlying, subchondral bone (e.g., bisphosphonates orosteoprotegerin (OPG)).

[0088] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivewithout interruption, but rather is cyclic in nature.

[0089] As used herein, “mammal” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic, and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, cats,sheep, pigs, cows, etc. The preferred mammal herein is a human. The term“non-adult” refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

[0090] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0091] “Carriers” as used herein include pharmaceutically-acceptablecarriers, excipients, or stabilizers that are non-toxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically-acceptable carrier is an aqueous pH-bufferedsolution. Examples of physiologically-acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low-molecular-weight (less thanabout 10 residues) polypeptides; proteins such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter-ions such as sodium; hyaluronan; and/or non-ionicsurfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0092] A “liposome” is a small vesicle composed of various types oflipids, phospholipids, and/or surfactants that is useful for delivery ofa drug (such as the IGF-1 analog or IGFBP displacer peptide disclosedherein) to a mammal. The components of the liposome are commonlyarranged in a bilayer formation, similar to the lipid arrangement ofbiological membranes.

[0093] The term “extended-release” or “sustained-release” formulationsin the broadest possible sense means a formulation of active IGF-1analog or IGFBP displacer peptide identified herein resulting in therelease or activation of the active analog or peptide for a sustained orextended period of time—or at least for a period of time that is longerthan if the analog or peptide were made available in vivo in the nativeor unformulated state. Optionally, the extended-release formulationoccurs at a constant rate and/or results in sustained and/or continuousconcentration of the active agent herein. Suitable extended-releaseformulations may comprise microencapsulation, semi-permeable matrices ofsolid hydrophobic polymers, biogradable polymers, biodegradablehydrogels, suspensions, or emulsions (e.g., oil-in-water orwater-in-oil). Optionally, the extended-release formulation comprisespoly-lactic-co-glycolic acid (PLGA) and can be prepared as described inLewis, “Controlled Release of Bioactive Agents form Lactide/Glycolidepolymer,” in Biodegradable Polymers as Drug Delivery Systems, M. Chasinand R. Langeer, Ed. (Marcel Dekker, New York), pp. 1-41. Optionally, theextended-release formulation is stable and the activity of the IGF-1analog or IGFBP displacer peptide as identified herein does notappreciably diminish with storage over time. More specifically, suchstability can be enhanced through the presence of a stabilizing agentsuch as a water-soluble polyvalent metal salt.

[0094] As used herein, “IGF-1” refers to insulin-like growth factor-1from any species, including bovine, ovine, porcine, equine, and human,preferably human, and, if referring to exogenous administration, fromany source, whether natural, synthetic, or recombinant.“Native-sequence” human IGF-1, the sequence of which is shown in FIG. 16(SEQ ID NO:3), is prepared, e.g., by the process described in EP 230,869published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; or EP288,451 published Oct. 26, 1988. More preferably, this native-sequenceIGF-1 is recombinantly produced.

[0095] As used herein, “IGF-2” refers to insulin-like growth factor-2from any species, including bovine, ovine, porcine, equine, and human,preferably human, and, if referring to exogenous administration, fromany source, whether natural, synthetic, or recombinant. It may beprepared by the method described in, e.g., EP 128,733.

[0096] An “IGFBP” or an “IGF binding protein” refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-1 orIGF-2, whether or not it is circulatory (i.e., in serum or tissue). Suchbinding proteins do not include receptors. This definition includesIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7),and prostacyclin-stimulating factor (PSF) or endothelial cell-specificmolecule (ESM-1), as well as other proteins with high homology toIGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc.Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol.Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al.,Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalleet al., J. Biol. Chem., 271: 20458-20464 (1996). For other identifiedIGFBPs, see, e.g., EP 375,438 published Jun. 27, 1990; EP 369,943published May 23, 1990; WO 89/09268 published Oct. 5, 1989; Wood et al.,Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBOJ., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411(1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 publishedDec. 7, 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al.,Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261:8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235(1988); WO 89/08667 published Sep. 21, 1989; WO 89/09792 published Oct.19, 1989; and Binkert et al., EMBO J., 8: 2497-2502 (1989).

[0097] The term “acid-labile subunit” or “ALS” refers to an 85-kDaglycoprotein that forms a ternary complex with IGF-1 and IGFBP-3 orIGFBP-5. See, e.g., Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995);Clemmons, Cytokine Growth Factor Rev., 8: 45-62 (1997); and Jones andClemmons, Endocr. Rev., 16: 3-34 (1995).

[0098] II. Modes for Carrying Out the Invention

[0099] The invention herein relates to the use of an IGF-1 analog or anIGFBP displacer peptide as defined above to treat cartilage disorders,preferably degenerative cartilagenous disorders, including regeneratingand/or preventing the degradation of cartilage.

[0100] Examples of IGF-1 analogs with a binding affinity preference forIGFBP-3 over IGFBP-1 include an IGF-1 variant wherein the amino acid(s)of wild-type human IGF-1 at position 3, 7, 10, 16, 25, or 49 or atpositions 3 and 49 of native-sequence human IGF-1 are replaced with analanine, a glycine, and/or a serine residue. Preferably, one or both ofthe amino acids in question are substituted by an alanine or glycineresidue, most preferably alanine. The more preferred IGF-1 analog withsuch binding affinity preference herein is F49A, F49G, F49S, E3A, E3G,E3S, E3AF49A, E3AF49G, E3AF49S, E3GF49A, E3GF49G, E3GF49S, E3SF49A,E3SF49G, E3SF49S, F16A, F16G, F16S, F16AF49A, F16GF49A, F16SF49A,F16AF49S, F16AF49G, F16SF49S, F16SF49G, F16GF49S, or F16GF49G.

[0101] Examples of IGF-1 analogs with a binding affinity preference forIGFBP-1 over IGFBP-3 include an IGF-1 variant wherein the amino acid(s)of wild-type human IGF-1 at position 9, 12, 15, or 20 is/are replacedwith a lysine or arginine residue. The more preferred IGF-1 analog withsuch binding affinity preference herein is D12K or D12R.

[0102] Examples of IGFBP-3 displacer peptides include a peptide selectedfrom the group consisting of:

[0103] Y24LY31A (IGF-1 variant); 4D3.3P (ASEEVCWPVAEWYLCNMWGR); (SEQ IDNO:6) BP3-4D3.11 (VAWEVCWDRHDQGYICTTDS); (SEQ ID NO:7) BP3-4D3.11_(DEL)(AWEVCWDRHQGYICTTDS); (SEQ ID NO:8) BP3-4B3.3 (EESECFEGPGYVICGLVG); (SEQID NO:9) BP3-01-ox (SEEVCWPVAEWYLCNMWG); (SEQ ID NO:10) BP3-02-ox(DMGVCADGPWMYVCEWTE); (SEQ ID NO:11) BP3-06 (TGVDCQC*GPVHC*VCMDWA); (SEQID NO:12) BP3-08 (TVANCDC*YMPLC*LCYDSD); (SEQ ID NO:13) BP3-15(SEEVCWPVAEWYLCN); (SEQ ID NO:14) BP3-16 (VCWPVAEWYLCNMWG); (SEQ IDNO:15) BP3-17 (VCWPVAEWYLCN); (SEQ ID NO:16) BP3-25 (CWPVAEWYLCN); (SEQID NO:17) BP3-27 (EVCWPVAEWYLCN); (SEQ ID NO:18) BP3-28(EEVCWPVAEWYLCN); (SEQ ID NO:19) BP3-30 (ASEEVCWPVAEWYLCN); (SEQ IDNO:20) BP3-39 (SEEVCWPVAEWYLCN-nh2); (SEQ ID NO:21) BP3-40(ac-SEEVCWPVAEWYLCN-nh2); (SEQ ID NO:22) BP3-41 (GPETCWPVAEWYLCN); (SEQID NO:23) BP3-107 (suc-CQLVRPDLLLCQ-nh2); and (SEQ ID NO:24) BP3-108(suc-IPVSPDWFVCQ-nh2); (SEQ ID NO:25)

[0104] where the C* indicates a cysteine that has been linked to anothercysteine in the peptide. The remaining Cys pairs are also oxidized asdisulfides in each peptide. The more preferred IGFBP-3 displacer peptideherein is BP3-15, BP3-39, BP3-40, BP3-01-OX, BP3-27, BP3-28, BP3-30,BP3-41, or 4D3.3P. The most preferred IGFBP-3 displacer peptide hereinis BP3-15, BP3-39, or BP3-40.

[0105] Examples of IGFBP-1 displacer peptides include a peptide selectedfrom the group consisting of: BP1-01 (CRAGPLQWLCEKYFG); (SEQ ID NO:26)BP1-02 (SEVGCRAGPLQWLCEKYFG; (SEQ ID NO:27) BP1-04 (CRAGPLQWLCE); (SEQID NO:28) BP1-10 (CRKGPLQWLCELYF); (SEQ ID NO:29) BP1-11(CRKGPLQWLCEKYF); (SEQ ID NO:30) BP1-12 (CKEGPLQWLCEKYF); (SEQ ID NO:31)BP1-13 (CKEGPLLWLCEKYF); (SEQ ID NO:32) BP1-14(SEVGCRAGPLQWLCEKYFG-nh2); (SEQ ID NO:33) BP1-15 (CAAGPLQWLCEKYF); (SEQID NO:34) BP1-16 (CRAGPLQWLCEKYF-nh2); (SEQ ID NO:35) BP1-17(CRAGPLQWLCEK-nh2); (SEQ ID NO:36) BP1-18 (CRAGPLQWLCEKAA); (SEQ IDNO:37) BP1-19 (SEMVCRAGPLQWLCEIYF-nh2*); (SEQ ID NO:38) BP1-20(EARVCRAGPLQWLCEKYF-nh2); (SEQ ID NO:39) BP1-21A(SEVGCRAGPLQWLCEKYFSTY-nh2); (SEQ ID NO:40) BP1-21B(CRAGPLQWLCEKYFSTY-nh2); (SEQ ID NO:41) BP1-25 (EARVCRAGPLQWLCEKYFSTY);(SEQ ID NO:42) BP1-40 (GQQSCRAGPLQWLCEKYFSTY); (SEQ ID NO:43) BP67(CRAGPLQWLCERYF); (SEQ ID NO:44) BP68 (CRAGPLQWLCEKFF); (SEQ ID NO:45)BP1-625 (GQQSCAAGPLQWLCEHYFSTYGR); (SEQ ID NO:46) BP1-625-Z(GQQSCAAGPLQWLCEHYFSTYGRGGGSGGAQHDEAVDNKFNKE (SEQ ID NO:47)QQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLN DAQAPNVDMN); BP1-625T(GQQSCAAGPLQWLCEHYFSTY); (SEQ ID NO:153) BP1027 (CKAGPLLWLCERFF); (SEQID NO:48) BP1028 (CRAGPLQWLCERFF); (SEQ ID NO:49) BP1029(CREGPLQWLCERFF); (SEQ ID NO:50) BP1030 (CKEGPLLWLCERFF); (SEQ ID NO:51)(i + 7)D (acRAGPLEWLAEKYEG); (SEQ ID NO:52) (i + 8)B (acRPLEWLAEKYFE);and (SEQ ID NO:53) (i + 8)C (acRAGPLEWLAEKYFE); (SEQ ID NO:54)

[0106] where the C* indicates a cysteine that has been linked to anothercysteine in the peptide, and the remaining Cys pairs are also oxidizedas disulfides in each peptide. The more preferred IGFBP-1 displacerpeptide herein is BP1-16, BP1-20, BP1-21A, BP1-25, BP1-40, BP625,BP625-Z, and BP625T; and most preferred are BP1-20, BP1-21A, BP1-25,BP1-40, BP1-625, BP1-625-Z, and BP1-625T.

[0107] The still more preferred active agents herein are F49A, E3A,F16A, E3AF49A, F16AF49A, D12K, D12R, BP3-15, BP3-40, BP3-39, BP1-16,BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, and BP1-625-Z; and the mostpreferred are F49A, E3AF49A, F16AF49A, D12K, D12R, BP3-15, BP3-40,BP3-39, BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, BP1-625-Z, andBP1-625T.

[0108] The IGF-1 analogs and IGFBP displacer peptides useful inaccordance with this invention can be made by any means that are knownin the art, including chemical synthesis or recombinant production.Chemical synthesis, especially solid phase synthesis, is preferred forshort (e.g., less than 50 residues) peptides or those containingunnatural or unusual amino acids such as D-Tyr, Ornithine, amino adipicacid, and the like. Recombinant procedures are preferred for longerpolypeptides. When recombinant procedures are selected, a synthetic genemay be constructed de novo or a natural gene may be mutated by, forexample, cassette mutagenesis. Set forth below are exemplary generalrecombinant procedures.

[0109] From a purified IGF-1 and its amino acid sequence, for example,an IGF variant that is a peptidyl mutant of an IGF-1 parent molecule maybe produced using recombinant DNA techniques. These techniquescontemplate, in simplified form, taking the gene, either natural orsynthetic, encoding the analog; inserting it into an appropriate vector;inserting the vector into an appropriate host cell; culturing the hostcell to cause expression of the gene; and recovering or isolating theanalog produced thereby. Preferably, the recovered analog is thenpurified to a suitable degree.

[0110] Somewhat more particularly, the DNA sequence encoding a peptidylIGF variant is cloned and manipulated so that it may be expressed in aconvenient host. DNA encoding parent polypeptides can be obtained from agenomic library, from cDNA derived from mRNA from cells expressing theparent polypeptide, or by synthetically constructing the DNA sequence(Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed.), ColdSpring Harbor Laboratory, N.Y., 1989).

[0111] The parent DNA is then inserted into an appropriate plasmid orvector which is used to transform a host cell. In general, plasmidvectors containing replication and control sequences that are derivedfrom species compatible with the host cell are used in connection withthose hosts. The vector ordinarily carries a replication site, as wellas sequences which encode proteins or peptides that are capable ofproviding phenotypic selection in transformed cells.

[0112] For example, E. coli may be transformed using pBR322, a plasmidderived from an E. coli species (Mandel et al., J. Mol. Biol. 53: 154(1970)). Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-lambda represent expression vectors with the tac, trp, or PLpromoters that are currently available (Pharmacia Biotechnology).

[0113] A preferred vector is pB0475. This vector contains origins ofreplication for phage and E. coli that allow it to be shuttled betweensuch hosts, thereby facilitating both mutagenesis and expression(Cunningham et al., Science, 243: 1330-1336 (1989); U.S. Pat. No.5,580,723). Other preferred vectors are pR1T5 and pR1T2T (PharmaciaBiotechnology). These vectors contain appropriate promoters followed bythe Z domain of protein A, allowing genes inserted into the vectors tobe expressed as fusion proteins.

[0114] Other preferred vectors can be constructed using standardtechniques by combining the relevant traits of the vectors describedabove. Relevant traits include the promoter, the ribosome binding site,the decorsin or ornatin gene or gene fusion (the Z domain of protein Aand decorsin or ornatin and its linker), the antibiotic resistancemarkers, and the appropriate origins of replication.

[0115] The host cell may be prokaryotic or eukaryotic. Prokaryotes arepreferred for cloning and expressing DNA sequences to produce the parentIGF-1 polypeptide, segment-substituted peptides, residue-substitutedpeptides, and peptide variants. For example, E. coli K12 strain 294(ATCC No. 31446) may be used as well as E. coli B, E. coli X1776 (ATCCNo. 31537), and E. coli c600 and c600hfl, E. coli W3110 (F-, gamma-,prototrophic/ATCC No. 27325), bacilli such as Bacillus subtilis, andother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species. The preferred prokaryote isE. coli W3110 (ATCC 27325). When expressed by prokaryotes, the analogsor peptides typically contain an N-terminal methionine or a formylmethionine and are not glycosylated. In the case of fusion proteins, theN-terminal methionine or formyl methionine resides on the amino terminusof the fusion protein or the signal sequence of the fusion protein.These examples are, of course, intended to be illustrative rather thanlimiting.

[0116] In addition to prokaryotes, eukaryotic organisms, such as yeastcultures, or cells derived from multicellular organisms may be used. Inprinciple, any such cell culture is workable. However, interest has beengreatest in vertebrate cells, and propagation of vertebrate cells inculture (tissue culture) has become a reproducible procedure. TissueCulture, Academic Press, Kruse and Patterson, editors (1973). Examplesof such useful host cell lines are VERO and HeLa cells, Chinese HamsterOvary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines.

[0117] A variation on the above procedures contemplates the use of genefusions, wherein the gene encoding the desired analog or peptide isassociated, in the vector, with a gene encoding another protein or afragment of another protein. This results in the desired analog orpeptide being produced by the host cell as a fusion with another proteinor peptide. The “other” protein or peptide is often a protein or peptidethat can be secreted by the cell, making it possible to isolate andpurify the desired analog or peptide from the culture medium andeliminating the necessity of destroying the host cells that arises whenthe desired analog or peptide remains inside the cell. Alternatively,the fusion protein can be expressed intracellularly. It is useful to usefusion proteins that are highly expressed.

[0118] The use of gene fusions, though not essential, can facilitate theexpression of heterologous analogs and peptides in E. coli as well asthe subsequent purification of those gene products (Harris, in GeneticEngineering, Williamson, R., Ed. (Academic Press, London, Vol. 4, 1983),p. 127; Ljungquist et al., Eur. J. Biochem., 186: 557-561 (1989) andLjungquist et al., Eur. J. Biochem., 186: 563-569 (1989)). Protein Afusions are often used because the binding of protein A, or morespecifically the Z domain of protein A, to IgG provides an “affinityhandle” for the purification of the fused protein. It has also beenshown that many heterologous proteins are degraded when expresseddirectly in E. coli, but are stable when expressed as fusion proteins(Marston, Biochem J., 240: 1 (1986)).

[0119] Fusion proteins can be cleaved using chemicals, such as cyanogenbromide, which cleaves at a methionine, or hydroxylamine, which cleavesbetween an Asn and Gly residue. Using standard recombinant DNAmethodology, the nucleotide base pairs encoding these amino acids may beinserted just prior to the 5′ end of the gene encoding the desiredanalog or peptide.

[0120] Alternatively, one can employ proteolytic cleavage of fusionprotein (Carter, in Protein Purification: From Molecular Mechanisms toLarge-Scale Processes, Ladisch et al., eds. (American Chemical SocietySymposium Series No. 427, 1990), Ch 13, pages 181-193).

[0121] Proteases such as Factor Xa, thrombin, and subtilisin or itsmutants, and a number of others have been successfully used to cleavefusion proteins. Typically, a peptide linker that is amenable tocleavage by the protease used is inserted between the “other” protein(e.g., the Z domain of protein A) and the desired analog or peptide.Using recombinant DNA methodology, the nucleotide base pairs encodingthe linker are inserted between the genes or gene fragments coding forthe other proteins. Proteolytic cleavage of the partially purifiedfusion protein containing the correct linker can then be carried out oneither the native fusion protein, or the reduced or denatured fusionprotein.

[0122] The analog or peptide may or may not be properly folded whenexpressed as a fusion protein. Also, the specific peptide linkercontaining the cleavage site may or may not be accessible to theprotease. These factors determine whether the fusion protein must bedenatured and refolded, and if so, whether these procedures are employedbefore or after cleavage.

[0123] When denaturing and refolding are needed, typically the analog orpeptide is treated with a chaotrope, such a guanidine HCl. Then it istreated with a redox buffer, containing, for example, reduced andoxidized dithiothreitol or glutathione at the appropriate ratios, pH,and temperature, such that the analog or peptide is refolded to itsnative structure.

[0124] When analogs and peptides are not prepared using recombinant DNAtechnology, they are preferably prepared using solid-phase synthesis,such as that generally described by Merrifield, J. Am. Chem. Soc., 85:2149 (1963), although other equivalent chemical syntheses known in theart are employable. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems peptidesynthesizer (Foster City, Calif.) following the manufacturer'sinstructions. Varous portions of the analog or peptide may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length analog or peptide.

[0125] Solid-phase synthesis is initiated from the C-terminus of theanalog or peptide by coupling a protected α-amino acid to a suitableresin. Such a starting material can be prepared by attaching anα-amino-protected amino acid by an ester linkage to a chloromethylatedresin or a hydroxymethyl resin, or by an amide bond to a BHA resin orMBHA resin. The preparation of the hydroxymethyl resin is described byBodansky et al., Chem. Ind. (London), 38: 1597-1598 (1966).Chloromethylated resins are commercially available from BioRadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., “Solid PhasePeptide Synthesis” (Freeman and Co., San Francisco 1969), Chapter 1, pp.1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired analog or peptide being synthesizedhas an unsubstituted amide at the C-terminus.

[0126] The amino acids are coupled to the analog/peptide chain usingtechniques well known in the art for the formation of peptide bonds. Onemethod involves converting the amino acid to a derivative that willrender the carboxyl group more susceptible to reaction with the freeN-terminal amino group of the peptide fragment. For example, the aminoacid can be converted to a mixed anhydride by reaction of a protectedamino acid with ethylchloroformate, phenyl chloroformate, sec-butylchloroformate, isobutyl chloroformate, pivaloyl chloride or like acidchlorides. Alternatively, the amino acid can be converted to an activeester such as a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester,a pentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole.

[0127] Another coupling method involves use of a suitable coupling agentsuch as N,N′-dicyclohexylcarbodiimide or N,N′-diisopropyl-carbodiimide.Other appropriate coupling agents, apparent to those skilled in the art,are disclosed in E. Gross and J. Meienhofer, The Peptides: Analysis,Structure, Biology, Vol. I: Major Methods of Peptide Bond Formation(Academic Press, New York, 1979).

[0128] It should be recognized that the α-amino group of each amino acidemployed in the analog/peptide synthesis must be protected during thecoupling reaction to prevent side reactions involving their activeα-amino function. It should also be recognized that certain amino acidscontain reactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, and hydroxyl) and that such functional groups must also beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at that site during both the initial and subsequentcoupling steps. Suitable protecting groups, known in the art, aredescribed in Gross and Meienhofer, The Peptides: Analysis, Structure,Biology, Vol.3: “Protection of Functional Groups in Peptide Synthesis”(Academic Press: New York, 1981).

[0129] In the selection of a particular side-chain protecting group tobe used in synthesizing the analogs/peptides, the following generalrules are followed. An α-amino protecting group (a) must render theα-amino function inert under the conditions employed in the couplingreaction, (b) must be readily removable after the coupling reactionunder conditions that will not remove side-chain protecting groups andwill not alter the structure of the analog/peptide fragment, and (c)must eliminate the possibility of racemization upon activationimmediately prior to coupling. A side-chain protecting group (a) mustrender the side chain functional group inert under the conditionsemployed in the coupling reaction, (b) must be stable under theconditions employed in removing the α-amino protecting group, and (c)must be readily removable upon completion of the desired analog/peptideunder reaction conditions that will not alter the structure of theanalog/peptide chain.

[0130] It will be apparent to those skilled in the art that theprotecting groups known to be useful for analog/peptide synthesis willvary in reactivity with the agents employed for their removal. Forexample, certain protecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, andp-methoxybenzyloxycarbonyl are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride inacetic acid, for their removal. Still other protecting groups, such asbenzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require stronger acids,such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetatein trifluoroacetic acid, for their removal. Among the classes of usefulamino acid protecting groups are included:

[0131] (1) for an α-amino group, (a) aromatic urethane-type protectinggroups, such as fluorenylmethyloxycarbonyl (FMOC) CBZ, and substitutedCBZ, such as, e.g., p-chlorobenzyloxycarbonyl,p-6-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, andp-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and thelike; (b) aliphatic urethane-type protecting groups, such as BOC,t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like;(c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;and d) allyloxycarbonyl. The preferred α-amino protecting groups are BOCor FMOC.

[0132] (2) for the side chain amino group present in Lys, protection maybe by any of the groups mentioned above in (1) such as BOC,p-chlorobenzyloxycarbonyl, etc.

[0133] (3) for the guanidino group of Arg, protection may be by nitro,tosyl, CBZ, adamantyloxycarbonyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC.

[0134] (4) for the hydroxyl group of Ser, Thr, or Tyr, protection maybe, for example, by C₁-C₄ alkyl, such as t-butyl; benzyl (BZL);substituted BZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl,o-chlorobenzyl, and 2,6-dichlorobenzyl.

[0135] (5) for the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using groups such as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

[0136] (6) for the imidazole nitrogen of His, the tosyl moiety issuitably employed.

[0137] (7) for the phenolic hydroxyl group of Tyr, a protecting groupsuch as tetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl,4-bromobenzyl, or 2,6-dichlorobenzyl is suitably employed. The preferredprotecting group is 2, 6-dichlorobenzyl.

[0138] (8) for the side-chain amino group of Asn or Gln, xanthyl (Xan)is preferably employed.

[0139] (9) for Met, the amino acid is preferably left unprotected.

[0140] (10) for the thio group of Cys, p-methoxybenzyl is typicallyemployed.

[0141] The C-terminal amino acid, e.g., Lys, is protected at the N-aminoposition by an appropriately selected protecting group, in the case ofLys, BOC. The BOC-Lys-OH can be first coupled to the benzyhydrylamine orchloromethylated resin according to the procedure set forth in Horiki etal., Chemistry Letters, 165-168 (1978) or using isopropylcarbodiimide atabout 25° C. for 2 hours with stirring. Following the coupling of theBOC-protected amino acid to the resin support, the α-amino protectinggroup is removed, as by using trifluoroacetic acid (TFA) in methylenechloride or TFA alone. The deprotection is carried out at a temperaturebetween about 0° C. and room temperature. Other standard cleavingreagents, such as HCl in dioxane, and conditions for removal of specifica-amino protecting groups are described in the literature.

[0142] After removal of the α-amino protecting group, the remainingα-amino and side-chain protected amino acids are coupled stepwise withinthe desired order. As an alternative to adding each amino acidseparately in the synthesis, some may be coupled to one another prior toaddition to the solid-phase synthesizer. The selection of an appropriatecoupling reagent is within the skill of the art. Particularly suitableas a coupling reagent is N,N′-dicyclohexyl carbodiimide ordiisopropylcarbodiimide.

[0143] Each protected amino acid or amino acid sequence is introducedinto the solid-phase reactor in excess, and the coupling is suitablycarried out in a medium of dimethylformamide (DMF) or CH₂Cl₂ or mixturesthereof. If incomplete coupling occurs, the coupling procedure isrepeated before removal of the N-amino protecting group prior to thecoupling of the next amino acid. The success of the coupling reaction ateach stage of the synthesis may be monitored. A preferred method ofmonitoring the synthesis is by the ninhydrin reaction, as described byKaiser et al., Anal. Biochem, 34: 595 (1970). The coupling reactions canbe performed automatically using well known methods, for example, aBIOSEARCH 9500™ peptide synthesizer.

[0144] Upon completion of the desired analog/peptide sequence, theprotected analog/peptide must be cleaved from the resin support, and allprotecting groups must be removed. The cleavage reaction and removal ofthe protecting groups is suitably accomplished simultaneously orstepwise. When the resin support is a chloro-methylated polystyreneresin, the bond anchoring the analog/peptide to the resin is an esterlinkage formed between the free carboxyl group of the C-terminal residueand one of the many chloromethyl groups present on the resin matrix. Itwill be appreciated that the anchoring bond can be cleaved by reagentsthat are known to be capable of breaking an ester linkage and ofpenetrating the resin matrix.

[0145] One especially convenient method is by treatment with liquidanhydrous hydrogen fluoride. This reagent not only will cleave theanalog/peptide from the resin but also will remove all protectinggroups. Hence, use of this reagent will directly afford the fullydeprotected analog/peptide. When the chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amines. Reaction withhydrogen fluoride in the presence of anisole and dimethylsulfide at 0°C. for one hour will simultaneously remove the side-chain protectinggroups and release the analog/peptide from the resin.

[0146] When it is desired to cleave the analog/peptide without removingprotecting groups, the protected analog/peptide-resin can undergomethanolysis to yield the protected analog/peptide in which theC-terminal carboxyl group is methylated. The methyl ester is thenhydrolyzed under mild alkaline conditions to give the free C-terminalcarboxyl group. The protecting groups on the analog/peptide chain thenare removed by treatment with a strong acid, such as liquid hydrogenfluoride. A particularly useful technique for methanolysis is that ofMoore et al., Peptides, Proc. Fifth Amer. Pept. Symp., M. Goodman and J.Meienhofer, Eds., (John Wiley, N.Y., 1977), p. 518-521, in which theprotected analog/peptide-resin is treated with methanol and potassiumcyanide in the presence of crown ether.

[0147] Another method for cleaving the protected analog/peptide from theresin when the chloromethylated resin is employed is by ammonolysis orby treatment with hydrazine. If desired, the resulting C-terminal amideor hydrazide can be hydrolyzed to the free C-terminal carboxyl moiety,and the protecting groups can be removed conventionally.

[0148] It will also be recognized that the protecting group present onthe N-terminal α-amino group may be removed preferentially either beforeor after the protected analog/peptide is cleaved from the support.

[0149] Purification of the analogs and peptides of the invention istypically achieved using conventional procedures such as preparativeHPLC (including reversed phase HPLC) or other known chromatographictechniques such as gel permeation, ion exchange, partitionchromatography, affinity chromatography (including monoclonal antibodycolumns) or countercurrent distribution.

[0150] The analogs and peptides of this invention may be stabilized bypolymerization. Polymerization may be accomplished by crosslinkingmonomer chains with polyfunctional crosslinking agents, either directlyor indirectly, through multi-functional polymers. Ordinarily, twosubstantially identical analogs/peptides are crosslinked at their C- orN-termini using a bifunctional crosslinking agent. The agent is used tocrosslink the terminal amino and/or carboxyl groups. Generally, bothterminal carboxyl groups or both terminal amino groups are crosslinkedto one another, although by selection of the appropriate crosslinkingagent the alpha-amino group of one analog/peptide is crosslinked to theterminal-carboxyl group of the other analog/peptide. Preferably, theanalogs/peptides are substituted at their C-termini with cysteine. Underconditions well known in the art a disulfide bond can be formed betweenthe terminal cysteines, thereby crosslinking the analog/peptide chains.For example, disulfide bridges are conveniently formed bymetal-catalyzed oxidation of the free cysteines or by nucleophilicsubstitution of a suitably modified cysteine residue. Selection of thecrosslinking agent will depend upon the identities of the reactive sidechains of the amino acids present in the analogs/peptides. For example,disulfide crosslinking would not be preferred if cysteine were presentin the analog/peptide at additional sites other than the C-terminus.Also within the scope hereof are analogs/peptides crosslinked withmethylene bridges.

[0151] Suitable crosslinking sites on the analogs/peptides, aside fromthe N-terminal amino and C-terminal carboxyl groups, include epsilonamino groups found on lysine residues, as well as amino, imino,carboxyl, sulfhydryl and hydroxyl groups located on the side chains ofinternal residues of the analogs/peptides or residues introduced intoflanking sequences. Crosslinking through externally added crosslinkingagents is suitably achieved, e.g., using any of a number of reagentsfamiliar to those skilled in the art, for example, via carbodiimidetreatment of the analog or peptide. Other examples of suitablemulti-functional (ordinarily bifunctional) crosslinking agents are foundin the literature.

[0152] The analogs and peptides of this invention also may beconformationally stabilized by cyclization. The analogs/peptidesordinarily are cyclized by covalently bonding the N- and C-terminaldomains of one analog/peptide to the corresponding domain of anotheranalog/peptide of this invention so as to form cyclo-oligomerscontaining two or more iterated analog/peptide sequences, each internalanalog/peptide having substantially the same sequence. Further, cyclizedanalogs/peptides (whether cyclo-oligomers or cyclo-monomers) arecrosslinked to form 1-3 cyclic structures having from 2 to 6 peptidescomprised therein. The analogs/peptides preferably are not covalentlybonded through α-amino and main chain carboxyl groups (head to tail),but rather are crosslinked through the side chains of residues locatedin the N and C-terminal domains. The linking sites thus generally willbe between the side chains of the residues.

[0153] Many suitable methods per se are known for preparing mono-orpoly-cyclized analogs/peptides as contemplated herein. Lys/Aspcyclization has been accomplished using Na-Boc-amino acids onsolid-phase support with Fmoc/9-fluorenylmethyl (OFm) side-chainprotection for Lys/Asp; the process is completed by piperidine treatmentfollowed by cyclization.

[0154] Glu and Lys side chains also have been crosslinked in preparingcyclic or bicyclic analogs/peptides: the analog/peptide is synthesizedby solid-phase chemistry on a p-methylbenzhydrylamine resin. Theanalog/peptide is cleaved from the resin and deprotected. The cyclicanalog/peptide is formed using diphenylphosphorylazide in dilutedmethylformamide. For an alternative procedure, see Schiller et al.,Peptide Protein Res., 25: 171-177 (1985). See also U.S. Pat. No.4,547,489.

[0155] Disulfide crosslinked or cyclized analogs/peptides are generatedby conventional methods. The method of Pelton et al. (J. Med. Chem., 29:2370-2375 (1986)) is suitable, except that a greater proportion ofcyclo-oligomers are produced by conducting the reaction in moreconcentrated solutions than the dilute reaction mixture described byPelton et al., for the production of cyclo-monomers. The same chemistryis useful for synthesis of dimers or cyclo-oligomers or cyclo-monomers.Also useful are thiomethylene bridges. Lebl and Hruby, TetrahedronLetters, 25: 2067-2068 (1984). See also Cody et al., J. Med. Chem., 28:583 (1985).

[0156] The desired cyclic or polymeric analogs/peptides are purified bygel filtration followed by reversed-phase high-pressure liquidchromatography or other conventional procedures. The analogs/peptidesare sterile filtered and formulated into conventional pharmacologicallyacceptable vehicles.

[0157] The starting materials required for the processes describedherein are known in the literature or can be prepared using knownmethods and known starting materials.

[0158] If in the analogs/peptides being created carbon atoms bonded tofour nonidentical substituents are asymmetric, then the analogs/peptidesmay exist as diastereoisomers, enantiomers or mixtures thereof. Thesyntheses described above may employ racemates, enantiomers ordiastereomers as starting materials or intermediates. Diastereomericproducts resulting from such syntheses may be separated bychromatographic or crystallization methods. Likewise, enantiomericproduct mixtures may be separated using the same techniques or by othermethods known in the art. Each of the asymmetric carbon atoms, whenpresent, may be in one of two configurations R) or S) and both arewithin the scope of the present invention.

[0159] The analogs and peptides of this invention may be contacted withthe cartilage by any suitable technique, and may be combined, analogwith analog, analog with peptide, or peptide with peptide. If treatmentis in vivo, the analog or peptide is administered to the mammal via,e.g., oral, parenteral (e.g., intramuscular, intraperitoneal,intravenous, intra-articular, or subcutaneous injection or infusion, orimplant), nasal, pulmonary, vaginal, rectal, sublingual, or topicalroutes of administration, and can be formulated in dosage formsappropriate for each route of administration. The specific route ofadministration will depend, e.g., on the medical history of the patient,including any perceived or anticipated side effects using the analog orpeptide, the type of analog or peptide being administered, and theparticular type of disorder to be corrected. Most preferably, theadministration is by continuous infusion (using, e.g., slow-releasedevices or minipumps such as osmotic pumps or skin patches), or byinjection (using, e.g., intravenous, intra-articular or subcutaneousmeans). Preferably, the analog or peptide is administered locally, forexample, directly to the joint where repair or prevention is needed.

[0160] The analog or peptide to be used in the therapy will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with the analog or peptide),the type of disorder, the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The effective amounts of the analog or peptidefor purposes herein are thus determined by such considerations and mustbe amounts that result in bioavailability of the drugs to the mammal andthe desired effect.

[0161] A preferred administration is a chronic administration of abouttwo times per day for 4-8 weeks to reproduce the effects of IGF-1. As analternative to injection, chronic infusion may be employed using aninfusion device for continuous subcutaneous (SC) or intra-articularinfusions. An intravenous bag solution may also be employed. The keyfactor in selecting an appropriate dose for the disorder in question isthe result obtained, as measured by criteria for measuring treatment ofthe cartilage disorder as are deemed appropriate by the medicalpractitioner.

[0162] As a general proposition, the total pharmaceutically-effectiveamount of the analog or peptide administered parenterally per dose willbe in a range that can be measured by a dose-response curve. Forexample, IGFs bound to IGFBPs or in the blood can be measured in bodyfluids of the mammal to be treated to determine the dosing.Alternatively, one can administer increasing amounts of the analog orpeptide to the patient and check the serum levels of the patient forIGF-1 and IGF-2. The amount of analog or peptide to be employed can becalculated on a molar basis based on these serum levels of IGF-1 andIGF-2.

[0163] Specifically, one method for determining appropriate dosing ofthe analog or peptide entails measuring IGF levels in a biological fluidsuch as a body or blood fluid. Measuring such levels can be done by anymeans, including RIA and ELISA. After measuring IGF levels, the fluid iscontacted with the analog or peptide using single or multiple doses.After this contacting step, the IGF levels are re-measured in the fluid.If the fluid IGF levels have fallen by an amount sufficient to producethe desired efficacy for which the molecule is to be administered, thenthe dose of the molecule can be adjusted to produce maximal efficacy.This method may be carried out in vitro or in vivo. Preferably, thismethod is carried out in vivo, i.e., after the fluid is extracted from amammal and the IGF levels measured, the analog or peptide herein isadministered to the mammal using single or multiple doses (that is, thecontacting step is achieved by administration to a mammal). Then the IGFlevels are re-measured from fluid extracted from the mammal.

[0164] Another method for determining dosing is to use antibodies to theanalog or peptide or another detection method for the analog or peptidein the LIFA format. This would allow detection of endogenous orexogenous IGFs bound to IGFBP and the amount of analog or peptide boundto the IGFBP.

[0165] Another method for determining dosing would be to measure thelevel of “free” or active IGF in blood. For some uses the level of“free” IGF would be a suitable marker of efficacy and effective doses ordosing. The amount of active IGF may also be measured in the synovialfluid.

[0166] For example, one method is described for detecting endogenous orexogenous IGF bound to an IGF binding protein or the amount of theanalog or peptide herein or detecting the level of unbound IGF in abiological fluid. This method comprises:

[0167] (a) contacting the fluid with 1) a means for detecting the analogor peptide that is specific for the analog or peptide (such as a firstantibody specific for epitopes on the analog or peptide) attached to asolid-phase carrier, such that in the presence of the analog or peptidethe IGF binding sites remain available on the analog or peptide forbinding to the IGF binding protein, thereby forming a complex betweenthe means and the IGF binding protein; and 2) the analog or peptide fora period of time sufficient to saturate all available IGF binding siteson the IGF binding protein, thereby forming a saturated complex;

[0168] (b) contacting the saturated complex with a detectably labeledsecond means which is specific for the IGF binding protein (such as asecond antibody specific for epitopes on the IGFBP) which are availablefor binding when the analog or peptide is bound to the IGF bindingprotein; and

[0169] (c) quantitatively analyzing the amount of the labeled meansbound as a measure of the IGFBP in the biological fluid, and thereforeas a measure of the amount of bound analog or peptide and IGF bindingprotein, bound IGF and IGF binding protein, or active IGF present in thefluid.

[0170] Given the above methods for determining dosages, in general, theamount of analog or peptide that may be employed can be estimated, i.e.,from about 1 μg/kg/day to 10 mg/kg/day, preferably about 10 μg/kg/day to1 mg/kg/day, more preferably about 10-200 μg/kg/day, might be used,based on kg of patient body weight, although, as noted above, this willbe subject to a great deal of therapeutic discretion.

[0171] It is noted that dosages and desired drug concentrations ofpharmaceutical compositions employable with the present invention mayvary depending on the particular use envisioned. The determination ofthe appropriate dosage or route of administration is well within theskill of an ordinary physician. Animal experiments provide reliableguidance for the determination of effective doses for human therapy.Interspecies scaling of effective doses can be performed following theprinciples laid down by Mordenti and Chappell, “The use of interspeciesscaling in toxicokinetics” in Toxicokinetics and New Drug Development,Yacobi et al., Eds., (Pergamon Press: New York, 1989), pp. 42-96.

[0172] The analog or peptide is suitably administered by asustained-release system. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman etal., Biopolymers, 22, 547-556 (1983), poly(2-hydroxyethyl methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer,Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include a liposomally entrappedanalog or peptide. Liposomes containing the analog or peptide areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (from or about 200 to 800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for themost efficacious therapy.

[0173] PEGylated analogs or peptides having a longer life can also beemployed, based on, e.g., the conjugate technology described in WO95/32003 published Nov. 30, 1995.

[0174] For parenteral administration, in one embodiment, the analog orpeptide is formulated generally by mixing each at the desired degree ofpurity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically, or parenterally, acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other peptides that are known to be deleterious topolypeptides.

[0175] Generally, the formulations are prepared by contacting the analogor peptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood or synovial fluid of the recipient. Examples of such carriervehicles include water, saline, Ringer's solution, a buffered solution,hyaluronan, and dextrose solution. Non-aqueous vehicles such as fixedoils and ethyl oleate are also useful herein.

[0176] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

[0177] The analog or peptide typically formulated in such vehicles at apH of from or about 4.5 to 8. It will be understood that use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of salts of the analog or peptide. The final preparation maybe a stable liquid or lyophilized solid.

[0178] Typically about 0.5 to 500 mg of the analog or peptide or mixtureof analogs and/or peptides, as the free acid or base form or as apharmaceutically acceptable salt, is compounded with a physiologicallyacceptable vehicle, carrier, excipient, binder, preservative,stabilizer, flavor, etc., as called for by accepted pharmaceuticalpractice. The amount of active ingredient in these compositions is suchthat a suitable dosage in the range indicated is obtained.

[0179] The analog or peptide to be used for therapeutic administrationmust be sterile. Sterility is readily accomplished by filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

[0180] The analog or peptide ordinarily will be stored in unit ormulti-dose containers, for example, sealed ampules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-mL vials are filled with 5mL of sterile-filtered 1% (w/v) aqueous solution of analog or peptide,and the resulting mixture is lyophilized. The infusion solution isprepared by reconstituting the lyophilized analog or peptide usingbacteriostatic Water-for-Injection.

[0181] Combination therapy with the analog or peptide herein and one ormore other appropriate reagents that enhance the effect of the analog orpeptide is also part of this invention. These include antagonists tocytokines, NO, or IL-1ra, a cartilage catabolism antagonist, or acartilage growth factor, such as wild-type IGF-1 and/or ALS if theactive agent is an IGFBP-3 displacer peptide or an IGF-1 analog with abinding affinity preference for IGFBP-3 over IGFBP-1. In addition, theIGFBP displacer peptide may be co-administered with an IGF-1 analogherein, preferably with the analog with a binding affinity preferencefor IGFBP-1 over IGFBP-3.

[0182] The active agent and reagent to enhance its effect may beadministered concurrently or sequentially, and the reagent may beadministered at the same or lower doses than they would otherwise beadministered if given alone. The displacer peptide/IGF-1 analog withbinding preference for IGFBP-3 can be administered separately from theIGF-1 and/or ALS, but preferably these agents are administered togetheras a binary or ternary complex where such a complex can be formed.Administration as a complex of ALS, IGF-1, and IGFBP-3 displacerpeptide/IGF-1 analog results in the longest half-life for the activeagent.

[0183] The invention herein also contemplates using gene therapy fortreating a mammal, using nucleic acid encoding the analog or peptide.Generally, gene therapy is used to increase (or overexpress) IGF levelsin the mammal. Nucleic acids that encode the analog or peptide can beused for this purpose. Once the amino acid sequence is known, one cangenerate several nucleic acid molecules using the degeneracy of thegenetic code, and select which to use for gene therapy.

[0184] There are two major approaches to getting the nucleic acid(optionally contained in a vector) into the patient's cells for purposesof gene therapy: in vivo and ex vivo. For in vivo delivery, the nucleicacid is injected directly into the patient, usually at the site wherethe analog or peptide is required. For ex vivo treatment, the patient'scells are removed, the nucleic acid is introduced into these isolatedcells and the modified cells are administered to the patient eitherdirectly or, for example, encapsulated within porous membranes which areimplanted into the patient. See, e.g., U.S. Pat. Nos. 4,892,538 and5,283,187.

[0185] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, viralinfection etc. A commonly used vector for ex vivo delivery of the geneis an adeno- or retro-virus.

[0186] The currently preferred in vivo nucleic acid transfer techniquesinclude infection with viral vectors (such as adenovirus, Herpes simplexI virus, retrovirus, or adeno-associated virus) and lipid-based systems(useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPEand DC-Chol, for example). In some situations it is desirable to providethe nucleic acid source with an agent that targets the target cells,such as an antibody specific for a cell surface membrane protein or thetarget cell, a ligand for a receptor on the target cell, etc. Whereliposomes are employed, proteins that bind to a cell-surface membraneprotein associated with endocytosis may be used for targeting and/or tofacilitate uptake. Examples include capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols, see Anderson et al., Science,256: 808-813 (1992). See also WO 93/25673 and the references citedtherein.

[0187] Kits are also contemplated for this invention. The article ofmanufacture comprises a container and an instruction. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition which is effectivefor treating the cartilage disorder, e.g., degenerative cartilagenousdisorder, and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The active agent in the compositionis an IGF-1 analog or an IGFBP displacer peptide as defined herein. Thecomposition can comprise any or multiple ingredients disclosed herein.The instruction on, or associated with, the container indicates that thecomposition is used for treating a cartilage disorder. For example, theinstruction could indicate that the composition is effective for thetreatment of osteoarthritis, rheumatoid arthritis, or any otherdegenerative cartilagenous disorder. The article of manufacture mayfurther comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. Alternatively, the compositionmay contain any of the carriers, excipients, and/or stabilizersmentioned hereinabove. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use. The kit optionally includes a separate container,preferably a vial, for a co-agent to be administered along with theactive agent, such as IGF-1.

[0188] The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLES

[0189] Examples 1-4 below are taken from WO 98/45427 as describingIGFBP-3 displacer peptides defined herein. Based upon the results of invitro and in vivo experiments using an IGFBP-3 displacer peptide withamino acid changes at residues 24 and 31 (Y24L, Y31A), also designated(Leu²⁴ Ala³¹) hIGF-1 or IGF-M, disclosed in WO 98/45427, it is predictedthat other peptides that inhibit the interaction of an IGF with anIGFBP, and bind poorly or not at all to the IGF-1 receptor, shouldincrease active IGF levels in a subject being treated. In addition, itis possible that another class of molecules might bind IGF-1 itself at asite remote from that involved in receptor interactions in such a way asto inhibit or prevent the interaction of IGF-1 with the IGFBPs, but notthe interaction of IGF-1 with its receptor.

[0190] In the examples, common α-amino acids may be described by thestandard one- or three-letter amino acid code when referring tointermediates and final products. By common α-amino acids is meant thoseamino acids incorporated into proteins under mRNA direction. Standardabbreviations are listed in The Merck Index, 10th Edition, ppMisc-2-Misc-3. Unless otherwise designated the common α-amino acids havethe natural or “L”-configuration at the alpha carbon atom. If the codeis preceded by a “D” this signifies the opposite enantiomer of thecommon α-amino acid. Modified or unusual α-amino acids such asnorleucine (Nle) and ornithine (Orn) are designated as described in U.S.Patent and Trademark Office Official Gazette 1114 TMOG, May 15, 1990.

Example 1 Phage-Derived Peptides to Bind IGF-1 and Binding Proteins

[0191] Introduction:

[0192] It has been shown that peptides that bind specifically and withmeasurable affinity to target molecules, such as proteins, can beidentified from an initial library of many binding and non-bindingpeptides through binding selections using bacteriophage coat-proteinfusions (Smith, Science, 228: 1315 (1985); Scott and Smith, Science,249: 386 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990); Devlin et al., Science, 249: 404 (1990); reviewed byWells and Lowman, Curr. Opin. Struct. Biol., 2: 597 (1992); U.S. Pat.No. 5,223,409). In addition, both proteins and peptides displayed onphage can be affinity-enhanced through iterative cycles of mutations,selection, and propagation.

[0193] Libraries of peptides differing in sequence at particular residuepositions can be constructed using synthetic oligodeoxynucleotides.Peptides are displayed as fusion proteins with a phage coat protein(such as g3p or g8p) on bacteriophage particles, each of which containsa single-stranded DNA genome encoding the particular peptide variant.After cycles of affinity purification, using an immobilized targetmolecule, individual bacteriophage clones are isolated, and the aminoacid sequence of their displayed peptides is deduced from their DNAsequences.

[0194] Materials and Methods:

[0195] Construction of Peptide-Phage Libraries

[0196] To identify a set of peptide molecules having the ability to bindto IGF-1 or to an IGF binding protein, such as IGFBP-1 or IGFBP-3,several diverse phage libraries of peptides, of length ranging from 18to 20 residues, were constructed. Peptides of this size were chosen inorder to favor the selection of peptides capable of maintainingwell-defined structures in solution. Because natural-amino acid peptidesof this size have a potential sequence diversity of 20¹⁸-20²⁰ (i.e.,2.6×10²³ to 1.0×10²⁶) variants, it is not practical to construct andtest all such variants. Instead, certain residues were fixed orconstant, which might be expected to allow or promote stable elements ofpeptide structure such as disulfide bonds or beta-turns, within eachpeptide.

[0197] Structural constraints or frameworks have previously been usedfor presentation of peptide libraries on phage and for subsequent,successive enhancement of binding affinities through mutation andselection. Such structured frameworks may favor stable bindingconformations of peptide segments. By analogy, immunoglobulins provide astable (and conserved) structural framework for presentation of adiversity of different peptide loops (CDR's, complementarity-determiningregions) which can bind different antigens.

[0198] Used as a template for library constructions was a plasmid,pt4.g8 (complete DNA sequence shown in FIG. 1) expressing anantibody-recognizable (gD-tag) peptide fused to g8p of bacteriophageM13. This plasmid contains single-stranded and double-stranded originsof DNA replication. The phoA promoter and STII secretion-signalsequences are upstream of the gD peptide (underlined below), which isfollowed by a “linker” peptide (double underlined below), and then theg8p of bacteriophage M13:

[0199]SGTAMADPNRFRGKDLAGSPGGGSGGGAEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS (SEQ ID NO:55)

[0200] Several random-sequence peptide libraries (Table I) wereconstructed using single-stranded template-directed mutagenesis (Kunkelet al., Methods. Enzymol., 204:125 (1991)), with the oligonucleotidesdescribed below. TABLE I Large Naive Libraries for g8 Display Oligo-Library nucleotide no. Peptide motif SEQ ID NO A HL-300SGTACX₂GPX₄CSLAGSP (SEQ ID NO:56) B HL-301 X₄CX₂GPX₄CX₄ (SEQ ID NO:57) CHL-302 X₂₀ (SEQ ID NO:58) D HL-303 X₇CX₄CX₇ (SEQ ID NO:59) D HL-304X₇CX₅CX₆ (SEQ ID NO:60) D HL-305 X₆CX₆CX₆ (SEQ ID NO:61) D HL-306X₆CX₇CX₅ (SEQ ID NO:62) D HL-307 X₅CX₈CX₅ (SEQ ID NO:63) D HL-308X₅CX₉CX₄ (SEQ ID NO:64) D HL-309 X₄CX₁₀CX₄ (SEQ ID NO:65)

[0201] A. Beta-Turn Sequence Motif

[0202] An example of a peptide of known three-dimensional structure isgiven by Wrighton et al., who selected a peptide agonist for theerythropoietin receptor (EPO-R) by phage display (Wrighton et al.,Science, 273: 458 (1996)). The peptide GGTYSCHFGPLTWVCKPQGG (SEQ IDNO:66) (having a disulfide bond joining the two Cys residues) forms adimer of two beta hairpins, in the crystallized complex with EPO-R(Livnah et al., Science, 273: 464 (1996)). Although the structure of theunbound form of this peptide in solution has not been reported, thebeta-turn structure formed by this peptide in complex with EPO-Rsuggested that similar structures might be formed by peptides of theform CX₂GPX₄C (SEQ ID NO:67).

[0203] As one type of structured peptide library, a portion of the gDpeptide was replaced with the motif CX₂GPX₄C (SEQ ID NO:67), leaving theupstream and downstream (“flanking”) residues unchanged from that of thestarting plasmid. Thus, this library was designed to display on phageparticles the peptide SGTACX₂GPX₄CSLAGSP (SEQ ID NO:56), where Xrepresents any of the 20 natural L-amino acids, fused to the linker andg8p described above. This library was constructed using theoligonucleotide HL-300:

[0204]5′-GCC TAT GCA TCT GGT ACC GCC TGC NNS NNS GGT CCT NNS NNS NNS NNSTGT TCT CTG GCA GGT TCA CCA G-3′ (SEQ ID NO:68),

[0205] where N indicates a mixture of the nucleotides A, G, C, and T,and S represents a mixture of the nucleotides G and C.

[0206] An additional library was constructed to allow for furtherinteractions within the peptide and/or with the target proteins byrandomizing the flanking sequences as well. This library was constructedwith the form X₄CX₂GPX₄CX₄ (SEQ ID NO:57) by using oligonucleotideHL-301:

[0207]5′-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS TGC NNS NNS GGT CCT NNSNNS NNS NNS TGT NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′ (SEQ IDNO:69).

[0208] B. Disulfide-Loop Motifs

[0209] Because many additional peptide conformations might be productivefor binding to a given target protein, it was desirable to test othertypes of peptide sequence motifs in phage-displayed libraries. Forexample, a single disulfide bond within a small peptide may favor stablestructures that allow for relatively higher-affinity binding than inunconstrained structures (Geysen et al., Mol. Immunol., 23: 709 (1986);Wood et al., Science, 232: 633 (1986); Oldenburg et al., Proc. Natl.Acad. Sci. USA, 89: 5393 (1992); O'Neil et al., Proteins, 14: 509(1992); McLafferty et al., Gene, 128: 29 (1993); Giebel et al.,Biochem., 34: 15430 (1995)). Several peptide-phage libraries weretherefore constructed, of the form X_(m)CX_(n)CX_(k), where m=4, n=10,and k=4, or where m=5, n=8-9, and k=4-5, or m=6, n=6-7, and k=5-6, orm=7, n=4-5, and k=6-7 (SEQ ID NOS:59 to 65). In these peptides, adisulfide bond is predicted to form a stabilizing constraint for peptideconformation.

[0210] These peptide libraries (see Table I) were constructed asX₇CX₄CX₇ (SEQ ID NO:59), using oligonucleotide HL-303: X₇CX₄CX₇, usingoligonucloetide HL-303: (SEQ ID NO:59) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS NNS NNS TGC NNS NNS NNS NNS (SEQ ID NO:70) TGC NNS NNSNNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′; X₇CX₅CX₆, usingoligonucleotide HL-304: (SEQ ID NO:60) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS NNS NNS TGC NNS NNS NNS NNS (SEQ ID NO:71) NNS TGC NNSNNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′; X₆CX₆CX₆, usingoligonucleotide HL-305: (SEQ ID NO:61) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS NNS TGC NNS NNS NNS NNS NNS (SEQ ID NO:72) NNS TGC NNSNNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′; X₆CX₇CX₅, usingoligonucleotide HL-306: (SEQ ID NO:62) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS NNS TGC NNS NNS NNS NNS NNS (SEQ ID NO:73) NNS NNS TGCNNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′; X₅CX₈CX₅, usingoligonucleotide HL-307: (SEQ ID NO:63) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS TGC NNS NNS NNS NNS NNS NNS (SEQ ID NO:74) NNS NNS TGCNNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′; X₅CX₉CX₄, usingoligonucleotide HL-308: (SEQ ID NO:64) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS NNS TGC NNS NNS NNS NNS NNS NNS (SEQ ID NO:75) NNS NNS NNSTGC NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA C-3′; and X₄CX₁₀CX₄, usingoligonucleotide HL-309: (SEQ ID NO:65) 5′-GCT ACA AAT GCC TAT GCA NNSNNS NNS NNS TGC NNS NNS NNS NNS NNS NNS NNS (SEQ ID NO:76) NNS NNS NNSTGC NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′.

[0211] C. Unconstrained Peptides

[0212] Unconstrained libraries (i.e., having no fixed residues withinthe peptide) have also yielded specific binding molecules (Scott andSmith, supra; Cwirla et al., supra; Devlin et al., supra; Kay et al.,Gene, 128: 59 (1993)). Such libraries may yield structured peptides,nevertheless, since noncovalent interactions may still induce structurein the bound and/or unbound forms. An unconstrained peptide library, ofthe form X₂₀ (SEQ ID NO:58), was constructed using oligonucleotideHL-302:

[0213] 5′-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS NNS NNS NNSNNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3′(SEQ ID NO:77).

[0214] Polyvalent (g8) Phage Binding Selections

[0215] The products of random mutagenesis reactions were transformedinto XL1-BLUE™ E. coli cells (Stratagene) by electroporation andamplified by growing 15-16 h with M13K07 (Vieira and Messing, MethodsEnzymol., 153: 3-11 (1987)) or VCSM13 helper phage (Stratagene Corp.).Based upon plating of the initial transformations, the number oftransformants per library was approximately 1.8×10⁸ for library HL-300,7.9×10⁸ for HL-301, 5.0×10⁸ for HL-302, 5.3×10⁸ for HL-303, 5.6×10⁸ forHL-304, 5.0×10⁸ for HL-305, 6.3×10⁸ for HL-306, 4.5×10⁸ for HL-307,1.9×10⁸ for HL-308, and 2.1×10⁸ for HL-309.

[0216] IGFBP-3 and IGF-1 were biotinylated with a 1.5:1 molar ratio of acleavable biotin reagent, EZ-LINK™ NHS-SS-Biotin (Pierce), to protein,using the manufacturer's instructions.

[0217] The initial selection of peptides for binding to IGFBP-3 or IGF-1was carried out using phage pools of approximately 1010 phage/ml (100 μltotal volume). MAXISORP™ 96-well plastic plates (Nunc) were coated witha solution of 2 ™g/ml of NEUTRAVIDIN™ brand avidin (Pierce) in 50 mMsodium carbonate buffer, pH 9.6, overnight at 4° C. The NEUTRAVIDIN™solution was then removed, and the plates were incubated with a blockingsolution of 5 g/l of bovine serum albumin, or 5 g/l of ovalbumin, or 5g/l of instant milk in 50 mM sodium carbonate buffer, for 1-2 h at roomtemperature. The blocking solution was then removed, and a solution ofbiotinylated target protein was added. After 1-2 h at room temperature,the target solution was removed, and the plates were washed ten timeswith PBS/TWEEN™ surfactant (0.05% TWEEN-20™ in PBS buffer).

[0218] Phage from the libraries described above were pooled as follows:pool A consisted of HL-300 phage, pool B of HL-301 phage, pool C ofHL-302 phage, and pool D of phage from the HL-303, HL-304, HL-305,HL-306, HL-307, HL-308, and HL-309 libraries. Phage were added inPBS/TWEEN™/albumin/biotin (PBS/TWEEN™ buffer with 1 μM biotin, 5 g/lbovine serum albumin, or ovalbumin) to wells coated with each target,and with control wells that were coated with NEUTRAVIDIN™ or withalbumin, but not biotinylated target. The phage were allowed to bind5-15 h at room temperature. The plates were then washed ten times withPBS/TWEEN™ buffer.

[0219] Phage remaining bound to the plates were eluted by incubatingwith 50 mM DTT for 1-2 h at room temperature. The eluted phage weretransfected into E. coli cells and allowed to grow overnight at 37° C.to amplify the phage.

[0220] The second and third cycles of binding selection were carried outas above, except that streptavidin (0.1 mg/ml) was included in the phagecocktails along with biotin. An aliquot was taken from eachtarget-coated and control well incubated with each library, and serialdilutions of the diluted phage were performed to measure specificbinding to target. The diluted phage were then transfected into E. colicells and plated for colony counting.

[0221] The fourth round of binding selection was carried out onMAXISORP™ plates directly coated with 2 μg/ml of each target protein, orwith albumin only. The results of phage-binding selections in cycles 2-4are shown in FIG. 2.

[0222] The same initial phage libraries (A, B, C, D) were also used forbinding selections to directly-coated IGFBP-3. In this case, MAXISORP™96-well plastic plates (Nunc) were coated with a solution of 2 μg/ml ofIGFBP-3 in 50 mM sodium carbonate buffer, pH 9.6, overnight at 4° C. Thetarget solution was then removed, and the plates were incubated with ablocking solution of 5 g/L of bovine serum albumin, for 1-2 h at roomtemperature. Phage were incubated with the plates as above, andnon-binding phage washed away. The phage remaining bound were eluted byincubating with 20 mM HCl for 10 min at room temperature. Thereafter,the acid-eluted phage were neutralized with one-fifth volume of 1 MTris-HCl, pH 8.0. Phage were transfected for colony counting asdescribed above.

[0223] Screening of Polyvalent Phage Clones (IGF-Blocking Phage assay)

[0224] Peptide-phage clones were isolated by mixing phage pools with E.coli cells, and plating onto antibiotic-containing media. Colonies wereisolated and grown with helper phage (as above) to obtainsingle-stranded DNA for sequencing. Peptide sequences selected forbinding IGFBP-3 or IGF-1 were deduced from the DNA sequences of phagemidclones. A number of such clones are represented by the peptide sequencesin Tables II and III, respectively. TABLE II Peptide sequences from g8display, IGFBP-3 selection Name Peptide sequence 4A3.1SGTACYGGPEWWCCSLAGSP (SEQ ID NO:78) 4A3.3 SGTACYGGPEWWCCSLAGSP (SEQ IDNO:79) 4A3.4 SGTACYGGPEWWCCSLAGSP (SEQ ID NO:80) 4B3.1DLAICAEGPEIWVCEETS (SEQ ID NO:81) 4B3.2 DFWICLSGPGWEECLEWW (SEQ IDNO:82) 4B3.3 EESECFEGPGYVICGLVG (SEQ ID NO:83) 4B3.4 DMGVCADGPWMYVCEWTE(SEQ ID NO:84) 4B3.5 DMGVCADGPWMYVCEWTE (SEQ ID NO:85) 4C3.1GSAGQGMTEEWAWIWEWWKE (SEQ ID NO:86) 4C3.2 ELDGWVCIKVGEQNLCYLAE (SEQ IDNO:87) 4C3.4 ELDGWVCIKVGEQNLCYLAE (SEQ ID NO:88) 4C3.4ELDGWVCIKVGEQNLCYLAE (SEQ ID NO:88) 4C3.5 ELDGWVCIKVGEQNLCYLAE (SEQ IDNO:88) 4D3.1 AIGGWCFIELDSLWCEEQIG (SEQ ID NO:89) 4D3.2SEDVECWQVWENLVCSVEHR (SEQ ID NO:90) 4D3.3 SEEVCWPVAEWYLCNMWGR (SEQ IDNO:91) 4D3.4 RVGAYISCSETECWVEDLLD (SEQ ID NO:92) 4D3.5WFKTVCYEWEDEVQCYTLEE (SEQ ID NO:93) 4D3.6 SEDVECWQVWENLVCSVEHR (SEQ IDNO:94) 4D3.7 RLEEQCVEVNYEPSCSFTAN (SEQ ID NO:95) 4D3.8SEEVCWPVAEWYLCNILGP (SEQ ID NO:96) 4D3.9 ETVANCDCYMDLCLCYGSDR (SEQ IDNO:97) 4D3.10 YHPISCMDHYYLIICDETVN (SEQ ID NO:98) 4D3.11VAWEVCWDRHDQGYICTTDS (SEQ ID NO:99) 4D3.12 AEWAECWIAGDQLLCVGKDN (SEQ IDNO:100) 23A3.1 EPWLCQYYEAAMLYLCWEEG (SEQ ID NO:101) 23A3.2AEEGMVWGWTGGWYNLDELC (SEQ ID NO:102) 23A3.3 SGGAIYWPVEQFIAFMAVGK (SEQ IDNO:103) 23A3.4 EPWLCQYYEAAMLYLCWEEG (SEQ ID NO:104) 23A3.5SGGAIYMPVEQFIAFMAVGK (SEQ ID NO:105) 23B3.1 TGVDCQCGPVHCVCMDWA (SEQ IDNO:12) 23B3.2 EVLLCSDGPQLYLCELYA (SEQ ID NO:106) 23B3.4SGVECVWGPQWGFCVEEY (SEQ ID NO:107) 23B3.5 DKEVCYLGPETWLCFWWP (SEQ IDNO:108) 23B3.6 EVLLCSDGPQLYLCELYA (SEQ ID NO:109) 23B3.7GDVECIEGPWGELCVWAD (SEQ ID NO:110) 23D3.1 FGGWSCQPTWVDVYVCNFEE (SEQ IDNO:111) 23D3.2 AMWVCVSDWETVEECIQYMY (SEQ ID NO:112) 23D3.3AMWVCVSDWETVEECIQYMY (SEQ ID NO:113) 23D3.4 AMWVCVSDWETVEECIQYMY (SEQ IDNO:114) 23D3.5 AMWVCVSDWETVEECIQYMY (SEQ ID NO:115) 23D3.6TNWFFVCESGHQDICWLAEE (SEQ ID NO:116)

[0225] TABLE III Peptide sequences from g8 display, IGF-1 selectionClone Peptide sequence Library Frequency HL-8 WVMECGAGPWPEGCTFML B 5/6(SEQ ID NO:117) HL-26 RKTSQGRGQEMCWETGGCS C 1/6 (SEQ ID NO:118) HL-25SWERGELTYMKLCEYMRLQQ C 4/6 (SEQ ID NO:119) HL-30 EHGRANCLITPEAGKLARVT C1/6 (SEQ ID NO:120)

[0226] Such peptide-phage clones could represent specific target-bindingpeptides which either do or do not block ligand (IGF-1 to IGFBP-3)binding, or any of a number of non-binding or background members of theselected pool. To distinguish among these possibilities, phage cloneswere tested for the ability to bind to IGFBP-3 in the presence andabsence of IGF-1.

[0227] IGFBP-3 was coated directly onto MAXISORP198 plates as above.Phage from clonal cultures were mixed with IGF-1 (100 nM finalconcentration), and incubated with the immobilized IGFBP-3 for 1 hour atroom temperature. The plates were then washed ten times, as above, and asolution of rabbit anti-phage antibody mixed with a goat-anti-rabbitconjugate of horseradish peroxidase was added. After an incubation of 1hour at room temperature, the plates were developed with a chromogenicsubstrate, o-phenylenediamine (Sigma). The reaction was stopped withaddition of ½ volume of 2.5 M H₂SO₄. Optical density at 490 nm wasmeasured on a spectrophotometric plate reader.

[0228] Titration of several IGFBP-3-selected peptide-phage clones showedall were inhibited by IGF-1 for binding to IGFBP-3 at some phageconcentration (FIGS. 3 and 4). These peptides are thus likely to occupyan overlapping site with the IGF-binding epitope on IGFBP-3. Additionalpeptide-phage clones were screened similarly, at a low concentration ofphage, with and without IGF-1.

[0229]FIG. 5 shows the results of a blocking assay of several phagemidclones derived from three rounds of DTT elution, followed by one roundof HCl elution, as described above. In each case, the phagemid clone wasgrown from a single colony overnight at 37° C. in a culture volume of 5ml. The phage particles were precipitated and resuspended in 0.5 ml ofPBS buffer. A 50-fold dilution of each phage solution was made intoPBS/TWEEN™ buffer, and the phage were incubated with or without 100 nMIGF-1 on an IGFBP-3-coated MAXISORP™ plate. As shown in FIG. 5, mostclones were >40% inhibited for binding to IGFBP-3 at these phageconcentrations, although clone 4D3.11 was only 5% inhibited under theseconditions.

[0230]FIG. 6 shows the results of a blocking assay of several phagemidclones derived from three rounds of HCl elution, as described above. Ineach case, the phagemid clone was grown from a single colony overnightat 37° C. in a culture volume of 5 ml. The phage particles were preparedas described above. In this case, as shown in FIG. 6, most cloneswere >80% inhibited for binding to IGFBP-3 at these phageconcentrations, although clones 23A3.3 and 23A3.5 were only about 20%inhibited under these conditions.

[0231] The variation in the degree to which phage binding is blocked bya constant concentration of IGF-1, as a function of phage dilution (FIG.3), or as a function of peptide displayed (FIGS. 5-6) is of interestbecause, without being limited to any one theory, it may be predictiveof (1) the degree of overlap between IGF-1- and peptide-binding epitopeson the IGFBP-3 molecule, and/or (2) the relative affinity of IGF-1versus phage-displayed peptide for binding to IGFBP-3. Since allpeptide-phage clones tested here showed some degree of inhibition withIGF-1, it is likely that the epitope for peptide-binding on IGFBP-3 foreach lies within an area occupied by bound IGF-1. Peptide assays (seebelow) support this conclusion (i.e., case 1). On the other hand,without being limited to any one theory, it is possible that somepeptide epitopes could be simply within an area for which binding of thephage particle displaying such peptides is sterically excluded by boundIGF-1.

[0232] The dependence of inhibition upon phage concentration, and thedifferences among phage clones (FIG. 3) may reflect case 2. Inparticular, phage clones whose binding to an IGFBP-3 coated plate wasinhibited only at low phage concentrations (e.g., 4D3.3, 4B3.4,corresponding to peptides BP3-01-ox and BP3-02-ox, respectively) appearto yield higher-affinity peptides (see below) for IGFBP-3 than do thosephage clones whose binding to an IGFBP-3 coated plate was inhibited bothat high and at low phage concentrations (e.g., 4C3.2, 4D3.5,corresponding to peptides BP-23 and BP-24, respectively).

[0233] Thus, this type of phage-titration blocking assay may begenerally useful as a means to predict the relative affinities andinhibitory potencies of peptides derived from phage displayed libraries.

[0234] Monovalent (g3) Display of IGFBP-3-Binding Peptides

[0235] Affinity maturation of a peptide or protein sequence bysuccessive rounds of random mutagenesis, selection, and propagation canbe efficiently accomplished when the copy number of displayed peptidesor proteins is limited (Bass et al., Proteins, 8: 309-314 (1990)). Suchan affinity maturation process is illustrated by the affinity maturationof hGH (U.S. Pat. No. 5,534,617). In this case, the copy number ofdisplayed hGH was limited by fusing the displayed protein to g3, ratherthan to g8 of bacteriophage particles, restricting the expression levelof hGH, and using a helper phage to supply wild-type g3p for phagemidpackaging and propagation.

[0236] To select for higher affinity peptide variants from pools ofphage displaying peptides on g8p, peptide cDNAs from two round 4 g8library pools, 4B and 4D, were transferred to a g3 vector for monovalentphage display. Binding selections were carried out for three rounds, asdescribed above, with acid elution of binding phage.

[0237] Peptide sequences obtained after three rounds of selections areshown in Table IV. Two clones, 4B3.3 and 4D3.11, dominated the selectedpools, and were seen in the earlier, g8 phage selections. A third clone,3Ai.2, represents a new peptide sequence that was not identified from g8display. In phage-ELISA competition assays, the apparent affinity of theg3-4B3.3 and g3-4D3.11 clones was <100 nM; however, the correspondingpeptides showed much weaker inhibition (see below). TABLE IV Peptidesequences from g3 display, IGFBP-3 selection Clone Peptide sequenceLibrary Frequency 3Ai.1 = EESECFEGPGYVICGLVG 4B  6/10 4B3.3 (SEQ IDNO:8) 3Ai.2 VEDECWMGPDWAVCWTWG 4B  4/10 (SEQ ID NO:121) 3Bi.1 =VAWEVCWDRHDQGYICTTDS 4D 10/10 4D3.11 (SEQ ID NO:6)

[0238] It is anticipated that affinity improvements can be obtained byiteratively mutating, selecting, and propagating peptide-phagelibraries, as described for hGH. See, e.g., U.S. Pat. No. 5,534,617.

[0239] Peptide Assays

[0240] Peptides were synthesized corresponding to a number ofphage-derived sequences. In cases where two Cys residues were found inthe peptide sequence, the disulfide (oxidized or “ox” suffix) monomericform of the peptide was prepared and purified. In cases where four Cysresidues were found, the {1-4,2-3}-disulfide form was prepared andpurified.

[0241] The ability of these peptides to bind IGFBP-3 and block IGF-1binding was tested in one or more of the following assays.

[0242] BIACORE™ Competition assay (for IGFBP-3 Binders)

[0243] IGF-1 was immobilized on a dextran chip for inhibition assaysusing a BIACORE™ 2000 surface-plasmon-resonance device (BIAcore, Inc.,Piscataway, N.J.) to measure free binding protein. IGF-1 wasbiotinylated as described above, and injected over a chip to whichstreptavidin had been coupled (BIAcore, Inc.) to give 400 to 800 RU(response units) of immobilized IGF-1. The IGF-1 showed no detectabledissociation over the time course of each experiment. Serial dilutionsof peptide were mixed with a constant concentration (40 nM) of IGFBP-3.After incubation for >1 hour at room temperature, an aliquot of 20 μLwas injected at a flow rate of 20 μL/min over the IGF-1 chip. Followingthe injection, a response reading was taken to measure the relativeamount of IGFBP-3 bound to the IGF-1.

[0244] The results (FIGS. 7-8) show a dose-response curve for eachpeptide's inhibition of IGFBP-3 binding to the chip. In particular, themost effective inhibitors of IGFBP-3 binding tested were peptidesBP3-01-ox (corresponding to phage clone 4D3.3), and a truncated form ofthis peptide, BP3-15 (see Table V). In that table, a disulfide bond isformed between the two Cys residues of each 2-Cys containing peptide.For peptides containing four cysteines, the two Cys* residues form adisulfide and the remaining two form a second disulfide. These peptidesshowed IC50's of 2 μM and 0.75 μM, respectively. Other peptides such asBP3-4D3.11 (phage clone 4D3.11 from g8 display and 3Bi.1 from g3display) showed inhibition with IC50's of <10 μM.

[0245] IGFBP-1 did not show binding to IGF-1 immobilized in this manner.TABLE V Inhibition of IGF-1 binding to IGFBP-3 by synthetic peptidesBIACORE ™ Peptide Sequence AssayIC50 (μM) BP-23ELDGWVCIKVGEQNLCYLAEG-nh2 220 (SEQ ID NO:122) BP-24WFKTVCYEWEDEVQCYTLEEG-nh2 100-300 (SEQ ID NO:123) BP-25RVGAYISCSETECWVEDLLDG-nh2 >1000 (SEQ ID NO:124) BP3-4D3.11VAWEVCWDRHDQGYICTTDS <10 (SEQ ID NO:7) BP3- AWEVCWDRHQGYICTTDS 804D3.11_(DEL) (SEQ ID NO:8) BP3-13 CWDRHDQGYICTTDS >1000 (SEQ ID NO:125)BP3-4B3.3 EESECFEGPGYVICGLVG 80 (SEQ ID NO:9) BP3-02-oxDMGVCADGPWMYVCEWTE 12 (SEQ ID NO:11) BP3-01-ox SEEVCWPVAEWYLCNMWG 2 (SEQID NO:10) BP3-15 SEEVCWPVAEWYLCN 0.75 (SEQ ID NO:14) BP3-16VCWPVAEWYLCNMWG 30 (SEQ ID NO:15) BP3-17 VCWPVAEWYLCN 9 (SEQ ID NO:16)BP3-06 TGVDCQC*GPVHC*VCMDWA 5 (SEQ ID NO:12) BP3-08 TVANCDC*YMPLC*LCYDSD15 (SEQ ID NO:13)

[0246] where nh2 means that the peptide has been blocked with an amideand where the C* indicates a cysteine that has been linked to anothercysteine in the peptide. The remaining Cys pairs are also oxidized asdisulfides in each peptide.

[0247] Radiolabeled IGF Assay (for IGFBP-3 Binders)

[0248] As an additional assay of peptide activity, several peptides weretested in an assay using ¹²⁵I-labeled IGF-1 to measure inhibition ofIGFBP binding, as described above (Assay 3). Serial dilutions of peptidewere added to an IGFBP-1 or an IGFBP-3 plate. Thereafter, ¹²⁵I-labeledIGF-1 was added and the plates were incubated for 2 hours. The plateswere then washed and counted to determine the amount of bound IGF-1.

[0249]FIG. 9 shows the inhibition of two IGFBP-3-selected peptides,BP3-01-ox and BP3-02-ox, for IGF-1 binding to an IGFBP-3 plate. Incontrast, these peptides did not inhibit IGF-1 binding to an IGFBP-1coated plate (FIG. 10).

[0250] In vitro Activation (KIRA)

[0251] The ability of several synthetic peptides to block IGF-1 bindingto IGFBPs and release functional IGF-1 was tested in a KIRA assay ofIGF-1 activity, as described above. Cells were treated with peptidealone, peptide plus IGF-1 plus IGFBP-1, peptide plus IGF-1, and peptideplus IGF-1 plus IGFBP-3. The results are shown in FIGS. 11A-D,respectively.

[0252] While BP3-01-ox has a lower affinity for the IGFBP than the othermolecules tested in this assay, the fact that BP3-01-ox inhibits bindingof IGFBP-3 to IGF-1 is, in itself, useful for various purposes,including for the LIFA and other assays noted above. Further, the KIRAassay only used IGF-1; it did not employ IGF-2, and BP3-01-ox was foundto inhibit binding of IGFBP-3 to IGF-2 as noted in the competition assaydescribed below.

[0253] BIACORE™ competition assay (for IGFBP-3 binders)

[0254] IGF-2 was immobilized on a dextran chip for inhibition assaysusing a BIACORE™ 2000 surface-plasmon-resonance device (BIAcore, Inc.,Piscataway, N.J.) to measure free binding protein. IGF-2 wasbiotinylated as described above, and injected over a chip to whichstreptavidin had been coupled (BIAcore, Inc.) to give approximately 1500RU of immobilized IGF-2. The IGF-2 showed no detectable dissociationover the time course of each experiment. Serial dilutions of peptidewere mixed with a constant concentration (20 nM) of IGFBP-3. Afterincubation for >1 hour at room temperature, an aliquot of 20 μl wasinjected at a flow rate of 20 μl/min over the IGF-2 chip. Following theinjection, a response reading was taken to measure the relative amountof IGFBP-3 bound to the IGF-2.

[0255] The results (e.g., see FIG. 12) show a dose-response curve foreach peptide's inhibition of IGFBP-3 binding to IGF-2. PeptidesBP3-01-ox, BP3-14, BP3-15, and BP3-17 showed IC50's of 0.92 μM, 1.0 μM,0.78 μM, and 5.1 μM, respectively. Thus, these peptides inhibit thebinding of IGFBP-3 both to IGF-1 and to IGF-2.

Example 2 Displacement of IGF-1 From IGFBPs Using BP3-15

[0256] This Example tests an IGFBP-3-specific peptide, BP3-15, for itsability to block the binding of ¹²⁵I-IGF-1 in human serum. Human serumwas incubated with ¹²⁵I-IGF-1 the peptide and the amount of tracer boundto IGFBPs via size-exclusion chromatography was measured. Addition ofthe peptide resulted in an approximate 42% decrease in ¹²⁵1-IGF-1associated with the 150-KD IGF/IGFBP-3/ALS complex and a 59% increase inthe amount of free ¹²⁵I-IGF-1. The peptide did not decrease ¹²⁵I-IGF-1binding to the 44-KD IGFBPs (in fact, it slightly increased it),indicating that the peptide only competes with IGF-1 for binding toIGFBP-3.

[0257] These results indicate that the analog (at 0.2 mM) can competewith IGF-1 for binding to IGFBP-3 in human serum.

Example 3 Relative Affinity of IGFBP-3 Binding Peptide Variants

[0258] The relative affinities of various BP3-01-ox variants weremeasured by the BIACORE™ competition assay. The results are shown inTable VI. It can be seen that 4D3.3P (SEQ ID NO:6), BP3-30 (SEQ IDNO:20), BP3-41 (SEQ ID NO:23), BP3-40 (SEQ ID NO:22), BP3-39 (SEQ IDNO:21), BP3-28 (SEQ ID NO:19), BP3-27 (SEQ ID NO:18), and BP3-25 (SEQ IDNO:17), have affinities similar to or greater than that of BP3-01-ox andare expected to increase the availability of IGF-1 in an in vitro cellculture assay. The lack of measurable activity for peptide BP3-24 (SEQID NO:126) indicates the critical role that the intact disulfide playsin maintaining a peptide conformation favorable for binding to IGFBP-3for this series of peptides. TABLE VI Relative affinities of BP3-01-oxvariants by BIACORE ™ competition assay IGF-1 Inhibition Variant PeptideIC50 IC50 (mut) / name sequence (μM) IC50 (wt) 4d3.3PASEEVCWPVAEWYLCNMWGR 5.6 2.8 (SEQ ID NO:6) BP3-30 ASEEVCWPVAEWYLCN 5.62.8 (SEQ ID NO:20) BP3-41 GPETCWPVAEWYLCN 4.0 2.0 (SEQ ID NO:23)BP3-01-ox SEEVCWPVAEWYLCNMWG 2.0 -1- (SEQ ID NO:10) BP3-40ac-SEEVCWPVAEWYLCN-nh2 0.66 0.33 (SEQ ID NO:22) BP3-39SEEVCWPVAEWYLCN-nh2 0.66 0.33 (SEQ ID NO:21) BP3-15 SEEVCWPVAEWYLCN 0.720.36 (SEQ ID NO:14) BP3-28 EEVCWPVAEWYLCN 5.4 2.7 (SEQ ID NO:19) BP3-27EVCWPVAEWYLCN 2.8 1.4 (SEQ ID NO:18) BP3-25 CWPVAEWYLCN 46 23 (SEQ IDNO:17) BP3-24 WPVAEWYLCN >1000 >500 (SEQ ID NO:126)

Example 4 Screening of Additional Libraries for Binding to IGFBP-3

[0259] Additional polyvalent (g8) peptide-phage libraries were desginedand sorted that yielded two peptides that inhibited IGFBP-3 binding toIGF-1. The results, shown in Table VII, indicate that BP3-107 (SEQ IDNO:24) and BP3-108 (SEQ ID NO:25) are inhibitors and they are expectedto increase the availability of IGF-1 in an in vitro cell culture assay.TABLE VII Peptide inhibition of IGFBP-3 binding to IGF-1 by BIACORE ™competition Peptide Phage parent Sequence IC50 (μM) BP3-107 t4H3.6suc-CQLVRPDLLLCQ-nh2 100 (SEQ ID NO:24) BP3-108 t4H3.9suc-IPVSPDWFVCQ-nh2  20 (SEQ ID NO:25)

Example 5 Structure/Function of BP1-01 and Affinity Maturation

[0260] A. Kinetics of BP1-01 Binding to IGFBP-1

[0261] WO 98/45427 published Oct. 15, 1998 discloses the preparation andcharacterization of the IGFBP-1 displacer peptide BP1-01(CRAGPLQWLCEKYFG) (SEQ ID NO:26). The kinetics of BP1-01 peptidevariants were examined in a BIAcore™ (BIAcore, Inc., Piscataway, N.J.)assay using IGFBP-1 covalently coupled via EDC/NHS (as described by themanufacturer) to a dextran chip. Peptide BP1-01 displayed dissociationkinetics too rapid to measure. However, BP1-02, the 19-mer variant(SEVGCRAGPLQWLCEKYFG) (SEQ ID NO:27) displayed measurable kinetics. Theassociation rate constant was 2.30×10⁵ M-1 sec⁻¹ and the dissociationrate constant was 5.03×10⁻² sec⁻¹. The latter implies a half-life forpeptide dissociation from IGFBP-1 of approximately 28 sec. Theassociation rate constant is moderately fast, consistent with the notionthat the peptide may not undergo significant conformation change uponbinding to IGFBP-1.

[0262] B. Scanning Mutagenesis of BP1-01 Peptides

[0263] Two series of synthetic peptide variants were generated todetermine which side chains of the BP1-01 peptide might contributedirectly to binding IGFBP-1. In the first series an alanine-scanningapproach (Cunningham and Wells, Science, 244: 1081-1085 (1989)) was usedto remove that portion of each side chain beyond the beta carbon. Thecontribution of these atoms to the free energy of binding of the peptideto IGFBP-1 was then assessed by measuring the potency (IC50) of thevariant for inhibiting IGFBP-1 binding to IGF-I or IGF-II in a BIAcorecompetition assay, analogous to that described for IGFBP-3. The resultsare shown in Table VIII.

[0264] A second series of peptides made use of non-natural amino acidsto probe whether other structural features such as an added methyl groupat the alpha carbon, or an isomer (D-alanine) could affect peptidebinding to IGFBP-1. The potencies of these peptides were measured bybiotinylated-IGFBP-1 ELISA assay, with the results shown in Table IX.These results confirm the importance of side chains L6, L9, W8, and Y13in the binding of BP1-01 to IGFBP-1. Structural contributions are alsosuggested by the effects of substitutions at R2 and A3.

[0265] In contrast, some substitutions, such as aib substitutions at G4,Q7, E11, K12, and F14, had little or no effect upon binding affinity.Peptides including one or more of these substitutions may neverthelessby useful because non-natural amino acids often confer upon a peptidegreater resistance to proteolysis (see Schumacher et al., Science, 271:1854 (1996) and references therein). Such peptides may achieve a longerhalf-life in serum than those having only natural amino acids.

[0266] In view of the results shown in Table IX, it is expected thatpeptides with a D-alanine substituted at position 2, 3, or 6 of BP1-01or with an alpha-aminoisobutyrate substituted at position 7, 8, 9, 11,12, 13, or 14 will increase the availability of IGF-I in an in vitrocell culture assay.

[0267] Lastly, the relative affinities of various C-terminal BP1-01variants were determined by ELISA, as shown in Table X. These data showthat the C-terminal region of the peptide is important for binding. Onlypeptide BP1-18 (SEQ ID NO:37) retained measurable inhibitory activityfor IGF-I:IGFBP-1 binding. It is expected that this peptide willincrease the availability of IGF-I in an in vitro cell culture assay.

[0268] Taken together, the structure-function data suggest that asmaller, including a non-peptidyl, compound could be designed to mimicthe action of the BP1-01 peptide by including elements of the C-terminusof this peptide in combination with the side chains L6, L9, W8, and Y13.TABLE VIII Relative affinities of BP1-01 Ala-scan peptide variants byBIAcore ™ IGF-I Inhibition IGF-II Inhibition Variant IC50 (mut) /IC50(wt) IC50 (mut) /IC50 (wt) C1 n.d. n.d. R2A 0.9 0.9 A3 -1- -1- G4 n.d.n.d. P5 n.d. n.d. L6A 30.3 34.7 Q7A 0.7 0.6 W8A 7.4 6.4 L9A 33.2 29.7C10 n.d. n.d. E11A 2.9 2.4 K12A 7.9 5.3 Y13A 12.5 14.6 F14A 6.2 5.8 (wt)-1- -1-

[0269] TABLE IX Relative affinities of BP1-01 non-natural peptidevariants by ELISA (a = D-alanine; aib = alpha-aminoisobutyrate) IGF-IInhibition Variant IC50 (mut)/IC50 (wt) C1 n.d. R2a 50 A3a 34 G4a 0.6 P5n.d. L6a 400 Q7aib 1.6 W8aib 24 L9aib 400 C10 n.d. E11aib 1.0 K12aib 2.0Y13aib 7.1 F14aib 3.0 (wt) -1-

[0270] TABLE X Relative affinities of C-terminal BP1-01 variants byELISA Variant Peptide IGF-I Inhibition name seq. IC50 (mut) /IC50 (wt)BP1-01 CRAGPLQWLCEKYFG -1- (SEQ ID NO:26) BP1-04 CRAGPLQWLCE >1000 (SEQID NO:28) BP1-17 CRAGPLQWLCEK >1000 (SEQ ID NO:36) BP1-18 CRAGPLQWLCEKAA148 (SEQ ID NO:37)

[0271] C. Polyvalent (g8) Selection of BP1-01 Secondary Libraries

[0272] NNS codons were used to generate diverse peptide libraries asdescribed above. Affinity selections were performed by solution bindingof phage to biotinylated IGFBP-1 (prepared as described above) insolution to minimize avidity effects. A similar strategy was used forantibody-phage selections by Hawkins et al., J. Mol. Biol., 226: 889(1992). For each round of selection, the target amount was reduced toselect for enhanced affinity variants. Typically, 10⁹-10¹⁰ purifiedphage were preblocked with MPBST (5% skim milk in PBS+0.05% TWEEN™ 20)for 1 hr at room temperature and screened for binding to biotinylatedtarget. Binding conditions are described below. Phage that bound totarget were captured by incubating with streptavidin-magnetic beads(Promega Corp., Madison, Wis.) for 2-5 minutes at room temperature.After binding, the beads were washed with PBS-TWEEN™/MPBST ten timesbefore eluting with 0.1 M HCl. The eluate was immediately neutralizedwith ⅓ volume of 1 M TRIS, pH 8.0. The eluted phage were propagated byinfecting XL1 for the next selection cycle. Rounds 1, 2, 3 were carriedout with 400 nM, 200 nM, and 20 nM target, respectively, with 1-hincubations. Round 4 was carried out with 4 nM target overnight. Allbinding reactions were performed at room temperature.

[0273] The identified mutations are shown in Table XIII of WO 98/45427and the relative affinities by ELISA plate assay or BIAcore™ are shownin Table XI below. It can be seen that BP1-10 (SEQ ID NO:29), BP1-11(SEQ ID NO:30), BP1-12 (SEQ ID NO:31), BP1-13 (SEQ ID NO:32), BP1-15(SEQ ID NO:34), BP68 (SEQ ID NO:45), BP1027 (SEQ ID NO:48), BP1028 (SEQID NO:49), BP1029 (SEQ ID NO:50), and BP1030 (SEQ ID NO:51) are ofcomparable or higher affinity than BP1-02 and BP1-01, and are thusexpected to increase the availability of IGF-I in an in vitro cellculture assay. TABLE XI Relative affinities of g8 BP1-01 selectants byELISA plate assay or BIAcore ™* Variant Peptide IGF-I Inhibition nameseq. IC50 (mut) /IC50 (wt) BP1-02 SEVGCRAGPLQWLCEKYFG 0.37 (SEQ IDNO:27) BP1-01 CRAGPLQWLCEKYFG -1- (SEQ ID NO:26) BP1-10 CRKGPLQWLCELYF1.1* (SEQ ID NO:29) BP1-11 CRKGPLQWLCEKYF 1.9* (SEQ ID NO:30) BP1-12CKEGPLQWLCEKYF 2.9* (SEQ ID NO:31) BP1-13 CKEGPLLWLCEKYF 2.5* (SEQ IDNO:32) BP1-14 SEVGCREGPLQWLCEKYF 0.26 (SEQ ID NO:33) BP1-15CAAGPLQWLCEKYF 0.68 (SEQ ID NO:34) BP67 CRAGPLQWLCERYF 0.34 (SEQ IDNO:44) BP68 CRAGPLQWLCEKFF 0.39 (SEQ ID NO:45) BP1027 CKAGPLLWLCERFF 8.8(SEQ ID NO:48) BP1028 CRAGPLQWLCERFF 4.6 (SEQ ID NO:49) BP1029CREGPLQWLCERFF 1.7 (SEQ ID NO:50) BP1030 CKEGPLLWLCERFF 4.3 (SEQ IDNO:51)

[0274] D. Monovalent (g3) Selection of BP1-01 Secondary Libraries

[0275] Monovalent (g3) selections of BP1-01 secondary libraries werecarried out essentially as described in part C above. Templatescontained either the TAA stop codon at the targeted sites forrandomization or an entirely unrelated binding sequence from BP1-01.Selection conditions were as described below with BSA replacing milk inthe blocking buffer. Phage-target complexes were captured by magneticstreptavidin beads (Promega Corp., Madison, Wis.). Biotinylated targetwas preincubated with phage for 1-3 h at room temperature in each round,with the target concentrations being reduced from 200-500 nM in round 1,to 50-100 nM in round 2, 10-50 nM in round 3, and 1-20 nM in round 4.

[0276] The identified mutations are shown in Table XV of WO 98/45427 andthe relative affinities, as determined by BIAcore competition assay orby ELISA plate assay (carried out as above, except that 5% acetonitrilewas used for peptide solubility) of several peptides selected are shownin Table XII below. BP1-16 (SEQ ID NO:35), a 13-residue version ofBP1-01 (lacking the C-terminal Gly), had similar affinity to that ofBP1-01. Substitutions at the N-terminus or C-terminus yielded affinityimprovements. For example, compared with BP1-16, addition of the STYsequence at the C-terminus yielded about a 3-fold affinity improvementfor peptide BP1-21B. (A similar effect was seen in the context of the18-mer: namely, a 3-fold improvement was observed between BP1-14 andBP1-21A. Substitution of the N-terminal S to G motif also improvedaffinity by 2- to 3-fold in peptides BP1-19 and BP1-20. All of thesepeptides had similar or improved apparent affinity for IGFBP-1 ascompared with BP1-01 and BP1-02 and are thus expected to increase theavailability of IGF-I in an in vitro cell culture assay. TABLE XIIRelative affinities of g3 BP1-01 selectants by BIAcore ™ or ELISA plateassay* IGF-I IGF-I Inhibi- Inhibi- tion tion IC50 IC50 (116)/ (114)/Variant Peptide C50 IC50 name seq. (mut) (mut) BP1-14SEVGCRAGPLQWLCEKYFG-nh2 4.8 -1- (SEQ ID NO:33) BP1-16 CRAGPLQWLCEKYF-nh2-1- 0.21 (SEQ ID NO:35) BP1-19 SEMVCRAGPLQWLCEIYF-nh2* 9.9 2.1 (SEQ IDNO:38) BP1-20 EARVCRAGPLQWLCEKYF-nh2 12 2.6 (SEQ ID NO:39) BP1-21ASEVGCRAGPLQWLCEKYFSTY-nh2 15 3.2 (SEQ ID NO:40) BP1-21BCRAGPLQWLCEKYFSTY-nh2 3.1 0.67 (SEQ ID NO:41)

Example 6 Alanine-Scanning Mutagenesis of IGF-1 and Structural IGF-1Analogs

[0277] Introduction:

[0278] An alanine-scanning mutagenesis approach (Cunningham and Wells,Science, 244: 1081-1085 (1989); U.S. Pat. No. 5,834,250) was used toremove that portion of each side chain of IGF-1 beyond the beta carbon.The contribution of these atoms to the free energy of binding of theIGF-1 analog to IGFBP-1 or to IGFBP-3 was then assessed by competitivephage ELISA. In this assay, IGFBP-1 or IGFBP-3 is used to inhibitIGF-phage mutants from binding to an IGFBP-1- or IGFBP-3-coatedimmunosorbant plate.

[0279] From a titration series of binding protein, binding (IC₅₀) can becalculated. Some mutants were also assessed for direct binding inBIACORE™ assays.

[0280] Materials and Methods:

[0281] Construction of Phagemid Vector and Mutagenesis

[0282] The gene encoding mature human IGF-1 was amplified from pBKIGF2B(U.S. Pat. No. 5,342,763) using PCR primers 5′-AGC TGC TTT GAT ATG CATCTC CCG AAA CTC TGT GCG GT-3′ (SEQ ID NO:127) and 5′-GAG CGA TCT GGG TCTAGA CAG ATT TAG CGG GTT TCA G-3′ (SEQ ID NO:128). The resulting fragmentwas cut with NsiI and XbaI, and ligated into pH 0753 previously digestedwith NsiI and XbaI. pH 0753 is a derivative of phGHam-g3 (Lowman et al.,Biochemistry, 30: 10832-10838 (1991)) in which the additional XbaI sitein the alkaline phosphatase promoter (PhoA) region has been deletedusing the oligonucleotide 5′-AAA AGG GTA TGT AGA GGT TGA GGT-3′ (SEQ IDNO:129). The ligated vector pH 0753 containing the IGF-1 open readingframe was named pIGF-g3. It encodes for IGF-1 harboring the doublemutation G15-A70V fused to a fragment of the gene III protein (residues249-406) from the E. coli bacteriophage M13. Binding of this IGF-1variant to IGFBP-1 and -3 was found to be indistinguishable fromwild-type IGF-1. Alanine mutagenesis was performed using single-strandedplasmid pIGF-g3 as template (Kunkel et al., Methods Enzymol., 204:125-139 (1991)). All residues of IGF-1 with the exception of cysteinesand alanines were singly replaced by alanine. The resulting constructswere verified by DNA sequencing.

[0283] Binding of IGF Mutants Displayed on Phage to IGFBP-1 and -3(Phage ELISA)

[0284] Immunosorbent plates (Nunc, MAXISORP™, 96 wells) were coated with100 μl/well of 1 μg/ml IGFBP-1 or IGFBP-3 in PBS buffer pH 7.2 at 4° C.overnight. The plates were then blocked with 0.5% TWEEN 20™/PBS (alsoused as binding buffer) for 2 hours at room temperature (proteinaceousblocking agents like bovine serum albumin were avoided to preventpotential IGF or IGFBP contamination). E. coli cells (XL1-Blue,Stratagene) freshly transformed with phagemid vector were grownovernight in 5 mL 2YT medium (Sambrook et al., supra) in the presence ofM13-VCS helper phage (Stratagene). Phage particles were harvested andresuspended in PBS buffer as described in Lowman, H. B., “Phage Displayof Peptide Libraries on Protein Scaffolds,” in Cabilly, S. (ed.),Combinatorial Peptide Library Protocols (Humana Press Inc.: Totowa,N.J., 1998), pp. 249-264. Then phage concentrations were normalized toyield a maximal ELISA signal of 0.2-0.4 for each mutant (Lowman, inCabilly, S. (ed.), supra). Threefold serial dilutions of solublecompetitor were prepared on non-absorbent microtiter plates (Nunc, F, 96wells) with binding buffer (0.5% TWEEN 20™/PB_(S)) containing phage atthe previously determined concentrations. The dilution range ofcompetitor protein extended over six orders of magnitude, starting at 5μM for IGFBP-1 and 500 nM for IGFBP-3. After blocking, the platescontaining immobilized target were washed with 0.05% TWEEN™/PBS bufferand subsequently incubated with 80 μl/well of the premixedphage-competitor solutions for 1 hour at room temperature. Afterwashing, bound phage was detected with 80 μl/well of a solutioncontaining a primary rabbit anti-phage polyclonal antibody and asecondary goat anti-rabbit monoclonal antibody-horseradish peroxidaseconjugate in 0.5% TWEEN 20™/PBS. o-Phenylenediamine (Sigma) andtetramethylbenzidine (Kirkegaard and Perry) were used as chromogenicsubstrates, resulting in product detection at 492 and 450 nm,respectively. IC₅₀ values were determined by fitting the binding data toa generic saturation curve (Lowman, in Cabilly, S. (ed.), supra). Atleast two individual clones of each IGF-1 mutant were assayed. Numbersin Table XIII represent mean±standard deviation of individually assessedIC₅₀ values. Expression and Purification of IGFBP-1 and IGFBP-3

[0285] Human IGFBP-1 was expressed in CHO cells and purified from theconditioned medium as described by Mortensen et al., Endocrinology, 138:2073-2080 (1997). Recombinant human IGFBP-3 has also been cloned andexpressed in mammalian cells (Wood et al., Mol. Endocrinology, 2:1176-1185 (1988)). Purification from conditioned medium essentiallyfollowed the procedure described for IGFBP-1, with use of an IGFaffinity column (Martin and Baxter, J. Biol. Chem., 261: 8754-8760(1986)).

[0286] Expression and Purification of Soluble IGF-1 Mutants

[0287] Plasmid pBKIGF2B (U.S. Pat. No. 5,342,763) expresses humanwild-type IGF-1 fused to the leader peptide of lamB under the control ofthe PphoA promoter. For ease of site-directed mutagenesis the phage florigin of replication (fl ori) was introduced into plasmid pBKIGF2B. Forthat purpose a 466-BP BamHI fragment containing the fl ori was excisedfrom pH 0753 (Lowman et al., supra, 1991), while plasmid pBKIGF2B waslinearized with EcoRI. Vector and fragment were both treated with Kienowenzyme to fill in restriction-site overhangs prior to blunt-endligation. Correct constructs were selected for the ability to producesingle-stranded phagemid DNA in the presence of M13VCS helper phage. Theresulting phagemid vector was named pBKIGF2B-fl-ori and was used astemplate to construct the IGF-1 ala-mutants of interest (see Table XIV)using the procedure of Kunkel et al., Methods Enzymol., 204: 125-139(1991)). Every mutagenesis step was confirmed by DNA sequencing.

[0288] Expression of IGF-1 mutants was as described for the IGF-1wild-type (Joly et al., Proc. Natl. Acad. Sci. USA, 95: 2773-2777(1998)), but without transient overexpression of oxidoreductases. Thepurification procedure was based on a previous protocol (Chang andSwartz, “Single-Step Solubilization and Folding of IGF-1 Aggregates fromEscherichia coli ” In Cleland, J. L. (ed.), Protein Folding In Vivo andIn Vitro (American Chemical Society, Washington, D.C., 1993), pp.178-188), with minor adaptations. Typically, 6 g of wet cell paste(equivalent to 2 liters low phosphate medium grown for 24 hrs) wasresuspended in 150 ml of 25 mM Tris-HCl pH 7.5 containing 5 mM EDTA.Cells were lysed in a microfluidizer (Microfluidics Corp., Newton,Mass.), and refractile particles containing accumulated IGF-1 aggregateswere collected by centrifugation at 12,000×g. Refractile particles werewashed twice with lysis buffer, twice with lysis buffer containing 1%N-lauroyl-sarcosine (Sigma) to extract membrane proteins, and twice withlysis buffer again. Washed refractile bodies were resuspended atapproximately 2 mg/ml in 50 mM CAPS(3-(cyclohexylamino)-1-propanesulfonic acid; Sigma) buffer pH 10.4containing 2 M urea, 100 mM NaCl, 20% MeOH, and 2 mM DTT. This procedurecombines solubilization of retractile bodies and subsequent oxidativerefolding of IGF-1 mutants (Chang and Swartz, supra). After 3 hrs atroom temperature the refolding solutions were filtered throughmicroconcentrator membranes (Centricon, Amicon) with a molecular weightcut off of 50 kDa. The majority of monomeric IGF-1 was recovered in theeluate, while higher molecular weight contaminants were concentrated inthe retentate. At this point IGF-1 fractions were >95% pure, as judgedfrom SDS-PAGE analysis. To separate correctly disulfide-bonded IGF-1from IGF-swap (containing two non-native disulfides; Hober et al.,Biochemistry, 31: 1749-1756 (1992); Miller et al., Biochemistry, 32:5203-5213 (1993)), refolding solutions were acidified with 5% aceticacid and loaded on a Dynamax™ C18 semi-preparative HPLC column (Varian;10.0 mm ID) at 4 ml/min. Buffers were H2 0/0.1% TFA (A) andacetonitrile/0.1% TFA (B). Separation of the disulfide isomers wasachieved by applying the following gradient: 0-30% B in 20 min, 30-45% Bin 60 min. The ratio of native IGF-1 to IGF-swap was usually about 2:1for each mutant, with IGF-swap eluting earlier in the gradient thannative IGF-1. The molecular mass of each mutant was verified by massspectrometry. After HPLC purification, samples were lyophilized andreconstituted at approximately 1 mg/ml in 100 mM HEPES buffer, pH 7.4.

[0289] Biosensor Kinetic Measurements

[0290] The binding affinities of the IGF variants for IGFBP-1 andIGFBP-3 were determined using a BIACORE™-2000 real time kineticinteraction analysis system (Biacore, Inc., Piscataway, N.J.) to measureassociation (k_(a)) and dissociation (k_(d)) rates. Carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) were activated with EDC(N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride) andNHS(N-hydroxysuccinimide) according to the supplier's instructions. Forimmobilization, IGF mutants in 20 mM sodium acetate, pH 4.8, wereinjected onto the biosensor chip at a concentration of 50 μg/ml to yieldapproximately 450-600 RU's (resonance-responce units) ofcovalently-coupled protein. Unreacted groups were blocked with aninjection of 1 M ethanolamine. Kinetic measurements were carried out byinjecting two-fold serial dilutions (starting at 1 μM) of either IGFBP-1or IGFBP-3 in running buffer (PBS, 0.05% TWEEN 20™, 0.1% ovalbumin, 0.1%sodium azide) at 25□C using a flow rate of 20 μl/min. Association rates(k_(a)) and dissociation rates (k_(d)) were calculated separately usinga 1:1 Langmuir™ association model in the BIACORE™ evaluation software v.3.0. The equilibrium dissociation constant (K_(D)) was calculated ask_(d)/k_(a).

[0291] Results:

[0292] Monovalent Phage Display of IGF-1

[0293] For a rapid and comprehensive alanine scan of the 70 amino acidresidues of IGF-1 it was first determined whether the protein could bemonovalently displayed on the surface of phage M13 (Bass et al., supra).Phage display technology combines the advantage of rapid single-strandedDNA mutagenesis with an easy purification of the resulting mutantprotein, simply by isolation of the corresponding phage particles (e.g.,Cunningham et al., EMBO J., 13: 2508-2515 (1994)). A vector wasconstructed in which mature human IGF-1 was fused to thecarboxy-terminal domain of the M13 gene III product. This constructincludes the stII signal sequence which directs the fusion protein tothe periplasmic space of E. coli and allows monovalent display of theprotein (Bass et al., supra; Lowman et al., supra, 1991). For cloningpurposes the first and the last amino acids of IGF-1 were changed; theresulting mutant G15-A70V was used as the template construct for thesubsequent alanine scanning mutagenesis.

[0294] When phage particles displaying IGF-1 G15-A70V were isolated andassayed in a binding competition phage ELISA for their affinity toIGFBP's, the IC₅₀ determined in that experiment were 8.5 nM for IGFBP-1and 0.5 nM for IGFBP-3 (FIG. 13). These values are in good agreementwith dissociation constants determined by BIACORE™ experiments usingwild-type IGF-1 (Heding et al., J. Biol. Chem., 271: 13948-13952(1996)). Wild-type IGF-1 affinities determined by radioactiveimmunoassays (RIA) are ˜2.8 nM for IGFBP-1 and ˜0.8 nM for IGFBP-3,further supporting the IC₅₀ values derived from phage ELISA.Additionally, phage particles displaying IGF-1 G15-A70V were efficientlycaptured by 11 independent monoclonal mouse anti-IGF-1 antibodiesimmobilized on microtiter plates. These results together suggested thatthe displayed IGF-variant is folded correctly and accessible on thesurface of the phage particles.

[0295] Ala-Scanning Mutagenesis of IGF-1 Binding to IGFBP-1 and IGFBP-3

[0296] All residues of G15-A70V IGF-1 with the exception of the fournative alanines and six cysteines were singly substituted by alanine,using the described G15-A70V IGF-1 gIII vector as a template.Additionally, the single mutants S1G and V70A and the double-mutationrestoring wild-type IGF-1 were constructed. Each of these constructs wasexpressed in E. coli and displayed on phage. IC₅₀ values for binding toIGFBP-1 and IGFBP-3 were determined by competitive phage ELISA as shownin FIG. 13. At least two different clones of every mutant were tested.The resulting IC₅₀ values are listed in Table XIII, and the loss or gainin IC₅₀ for each mutant with respect to G15-A70V is graphed in FIG. 14.TABLE XIII Apparent Affinities (IC₅₀) of IGF-l Variants for IGFBP-1 andIGFBP-3 Determined by Phage Display^(a) IGFBP-3 IGFBP-1 relative IGF-1Relative relative speci- mutant IC₅₀ (nM) IC₅₀ IC₅₀ (nM) IC₅₀ ficity S1A5.2 ± 0.9 0.6 0.91 ± 0.32 1.2 0.5 P2A 11.0 ± 3.7  1.3 0.81 ± 0.18 1.11.2 E3A  278 ± 86   33.9 1.05 ± 0.08 1.4 24.2 T4A 19.4 ± 6.4  2.4 0.80 ±0.02 1.1 2.2 L5A 55.3 ± 11.6 6.7 1.53 ± 0.22 2.0 3.3 G7A >1000 >100 4.58± 0.28 6.1 >16 E9A 8.6 ± 0.6 1.0 1.32 ± 0.30 1.8 0.6 L10A  311 ± 87  37.9 3.55 ± 0.33 4.7 8.1 V11A* n.d. — n.d. — — D12A 4.3 ± 0.8 0.5 1.49 ±0.38 2.0 0.3 L14A 36.7 ± 1.1  4.5 0.90 ± 0.04 1.2 3.7 Q15A 13.9 ± 0.9 1.7 1.26 ± 0.41 1.7 1.0 F16A 57.8 ± 20.1 7.0 1.32 ± 0.25 1.8 4.0 V17A42.9 ± 3.2  5.2 3.67 ± 1.02 4.9 1.1 G19A 11.0 ± 2.3  1.3 0.90 ± 0.28 1.21.1 D20A 8.4 ± 4.1 1.0 1.11 ± 0.06 1.5 0.7 R21A 7.1 ± 1.6 0.9 0.58 ±0.01 0.8 1.1 G22A 15.9 ± 2.8  1.9 2.07 ± 0.11 2.8 0.7 F23A 10.9 ± 1.9 1.3 2.18 ± 0.01 2.9 0.5 Y24A 13.3 ± 2.9  1.6 2.53 ± 0.76 3.4 0.5 F25A 181 ± 46   22.1 3.69 ± 0.25 4.9 4.5 N26A 9.1 ± 1.8 1.1 0.90 ± 0.07 1.20.9 K27A 12.8 ± 0.1  1.6 0.66 ± 0.35 0.9 1.8 P28A 9.3 ± 1.4 1.1 1.41 ±0.05 1.9 0.6 T29A 7.3 ± 2.4 0.9 1.23 ± 0.16 1.6 0.5 G30A 7.1 ± 1.7 0.90.58 ± 0.11 0.8 1.1 Y31A 6.8 ± 0.5 0.8 0.73 ± 0.10 1.0 0.9 G32A 10.9 ±1.3  1.3 0.76 ± 0.28 1.0 1.3 S33A 9.1 ± 1.0 1.1 1.01 ± 0.24 1.3 0.8 S34A9.5 ± 0.7 1.2 1.65 ± 0.21 2.2 0.5 S35A 11.7 ± 0.6  1.4 0.47 ± 0.01 0.62.3 R36A* n.d. — n.d. — — R37A 12.3 ± 0.1  1.5 0.75 ± 0.08 1.00 1.5P39A* n.d. — n.d. — — Q40A 10.2 ± 0.9  1.2 0.56 ± 0.03 0.7 1.7 T41A 13.7± 3.1  1.7 0.43 ± 0.06 0.6 2.9 G42A 15.7 ± 3.4  1.9 0.53 ± 0.20 0.7 2.7I43A 31.3 ± 4.1  3.8 1.17 ± 0.07 1.6 2.4 V44A 18.8 ± 5.4  2.3 1.03 ±0.06 1.4 1.7 D45A 4.7 ± 0.7 0.6 0.69 ± 0.21 0.9 0.6 E46A 7.9 ± 2.1 1.00.94 ± 0.28 1.3 0.8 F49A >1000 >100 2.72 ± 1.11 3.6 >28 R50A 16.2 ± 1.8 2.0 0.64 ± 0.18 0.9 2.3 S51A 13.4 ± 0.4  1.6 0.65 ± 0.35 0.9 1.9 D53A15.3 ± 2.8  1.9 1.05 ± 0.11 1.2 1.6 L54A 23.1 ± 12.0 2.8 1.83 ± 0.91 2.41.2 R55A 9.0 ± 2.3 1.1 0.66 ± 0.03 0.9 1.2 R56A 13.1 ± 1.8  1.6 1.00 ±0.19 1.3 1.2 L57A 21.8 ± 5.6  2.7 1.78 ± 0.56 2.4 1.1 E58A 11.9 ± 1.8 1.5 1.03 ± 0.47 1.4 1.1 M59A 13.1 ± 1.8  1.6 0.74 ± 0.14 1.0 1.6 Y60A6.6 ± 1.8 0.8 0.52 ± 0.01 0.7 1.2 P63A >1000 >100 >100 >100 — L64A 12.1± 3.3  1.5 0.93 ± 0.03 1.2 1.2 K65A 12.4 ± 0.6  1.5 0.69 ± 0.05 0.9 1.6P66A 9.4 ± 3.2 1.1 0.57 ± 0.12 0.8 1.5 K68A 10.5 ± 2.8  1.3 0.76 ± 0.231.0 1.3 S69A 12.8 ± 2.3  1.6 0.71 ± 0.62 1.2 1.3 V70A 19.1 ± 0.7  2.30.68 ± 0.15 0.9 2.6 S1G 11.2 ± 1.1  1.4 0.99 ± 0.42 1.3 1.0 IGF-1 WT 8.4± 0.8 1.0 1.01 ± 0.42 1.3 0.8 G1S-A70V 8.2 ± 1.6 1.0 0.75 ± 0.32 1.0 1.0Ala(1-3)-IGF 90.4 ± 9.6  11.0 1.12 ± 0.04 1.5 7.3 Des(1-2)-IGF 5.0 ± 0.10.6 0.53 ± 0.03 0.7 0.9

[0297] The majority of the alanine mutants yielded only minor changes inIC₅₀ values in the phage ELISA. Importantly, wild-type IGF-1 showed thesame affinities for IGFBP-1 and IGFBP-3 as G15-A70V in which backgroundthe alanine substitutions were performed (Table XIII, FIG. 14). Only afew residues caused considerable (>10-fold) losses in affinity whenchanged to alanine: E3, G7, L10, V11, F25, R36, P39, F49, and P63 forIGFBP-1 binding; V11, R36, P39, and P63 for IGFBP-3 binding. It has beennoted that ala-substitutions of glycines and prolines can lead tostructural perturbations of the protein backbone (Di Cera, Chem. Rev.,98: 1563-1591 (1998)).

[0298] Only a few modest improvements in binding affinity were found byalanine replacements. S1A, D12A, and D45A showed an approximately 2-foldincrease in IGFBP-1 binding, while S35A and T41A showed a similar effectfor IGFBP-3. However, 2-fold changes in IC₅₀ values are at the limit ofprecision in these experiments.

[0299] IGFBP-Specificity Determinants

[0300] E3A, G7A, L10A, F25A, and F49A showed a differential effect inbinding IGFBP-1 versus IGFBP-3. For these five IGF-1 single alaninemutants the relative IC₅₀ for IGFBP-1 differed by more than 4-fold fromthe one for IGFBP-3 (FIG. 14, Table XIII, relative specificity). E3A andF49A showed the biggest relative specificity factors in this group.Alanine substitution of E3 had virtually no effect on IGFBP-3 affinity(1.4 fold), while binding to IGFBP-1 was weakened 34-fold. Even moredramatic, the affinity of F49A was reduced more than 100-fold forIGFBP-1 but only 3.6-fold for BP-3. This result was illustrated in adirect comparison by phage ELISA. Phage particles displaying IGF-1 F49Awere added to IGFBP-3 coated wells in the presence of soluble IGFBP-1(FIG. 15A) or IGFBP-3 (FIG. 15B). Compared to control phage displayingIGF-1 G15-A70V, the binding curve of F49A shifted by more than twoorders of magnitude in the IGFBP-1 competition (FIG. 15A). In contrast,the binding curves were similar in the IGFBP-3 competition, and the IC₅₀values differed by less than a factor of 4 (FIG. 15B). Thus, E3 and F49are two major specificity determinants for IGFBP-1 binding in the IGF-1molecule.

[0301] Residues G7, L10, and F25 appeared to be important for binding ofboth IGFBP's, although showing a more pronounced loss of affinity forIGFBP-1 than for IGFBP-3 when substituted by alanines. No significantspecificity determinant for IGFBP-3 was identified, such as a mutantbinding much tighter to IGFBP-1 than to IGFBP-3. However, mutations E9A,D12A, F23A, Y24A, T29A, S34A, and D45A had slightly larger (about2-fold) effects on IGFBP-3 than on IGFBP-1 binding.

[0302] BIACORE™ Measurements of Purified Soluble IGF Mutants

[0303] For validation of the results obtained by phage ELISA, specificalanine mutants were expressed and purified for kinetic analysis using aBIACORE™ instrument. The dissociation constant (K_(D)) of wild-typeIGF-1 was determined to be 13 nM for IGFBP-1 and 1.5 nM for IGFBP-3(FIGS. 17A and 17B; Table XIV). The difference in affinity for theIGFBP's is due to a 10-fold faster association rate (k_(a)) of IGF-1 toIGFBP-3 (3.2×10⁵ versus 3.2×10⁴ M⁻¹s⁻¹). These results correspond wellwith the absolute IC₅₀ values determined by phage ELISA (FIGS. 13A and13B; Table XIII). As expected, the double-mutant G15-A70V showed kineticparameters essentially indistinguishable from wild-type (Table XIV).

[0304] V11A, R36A, and P39A were tested because these variants had notbeen displayed correctly on phage, based upon the antibody recognitionexperiments (see above). R36A and P39A showed wild-type kinetics forboth binding proteins, whereas V11A showed a 5-fold reduction inaffinity for both IGFBP-1 and IGFBP-3.

[0305] Furthermore, it was decided to examine the soluble IGF variantT4A. This residue had been implicated in IGFBP binding in earlierpublications (Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988);Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990)), but had shownmodest effects in the phage assays herein. The increase in the K_(D)values of T4A relative to wild-type IGF-1 was approximately 2-3-foldhigher than the IC₅₀ ratios determined by phage ELISA (Table XIV). Abigger discrepancy between the results obtained by phage and thebiosensor analysis was seen for F16A. In this case the two methodsdiffered by a factor of 4.

[0306] It has been shown that mutations in the first α-helical regionhave a destabilizing effect on the IGF-protein structure (Jansson etal., Biochemistry, 36: 4108-4117 (1997)). Without being limited to anyone theory, it is believed that the g3 fusion protein on the surface ofthe phage might be more stable than the refolded, purified solubleprotein. This is supported by the BIACORE™ results obtained for F25A andF49A, two residues located outside the structurally sensitive N-terminalhelix. The respective changes in K_(D) and IC₅₀ values are in excellentagreement for these two mutants (Table XIV). The differential effect ofF49A on binding to the IGFBP's was confirmed by the BIACORE™ analysis. A70-fold decrease in affinity was measured for IGFBP-1 binding (FIG. 17C;Table XIV), whereas IGFBP-3 binding was reduced only 4-fold (FIG. 17D;Table XIV). TABLE XIV Kinetic Parameters for the Interaction of PurifiedIGF-1 Variants with IGFBP-1 and -3 Determined by BIACORE ™ Analysis^(a)Binding to IGFBP-1 k_(a) k_(d) K_(D) (× 10⁴ M⁻¹s⁻¹) (× 10⁴ s⁻¹) (nM)relative K_(D) relative IC₅₀ IGF-1 WT 3.2 ± 0.2 4.1 ± 0.2 13.0 ± 1.0 1.0 1.0 G1S-A70V 3.2 ± 0.2  4.5 ± 0.01 14.0 ± 0.7  1.1 1.0 T4A 1.9 ± 0.216.7 ± 1.6  90.0 ± 11.0 6.9 2.4 V11A 1.9 ± 0.1 12.3 ± 0.6  66.5 ± 4.5 5.1 — F16A 1.9 ± 0.6 60.3 ± 4.5   321 ± 98   25 6.0 F25A 1.5 ± 0.5 49.0± 5.7   323 ± 107  25 22 R36A 4.0 ± 0.2 5.6 ± 0.2 13.9 ± 0.8  1.1 — P39A3.1 ± 0.2 4.2 ± 0.1 13.6 ± 0.8  1.0 — F49A 1.26 ± 0.8   115 ± 1.5   913± 551  70 >100 Binding to IGFBP-3 k_(a) k_(d) K_(D) (× 10⁵ M⁻¹s⁻¹) (×10⁴ s⁻¹) (nM) relative K_(D) relative IC₅₀ IGF-1 WT 3.2 ± 0.5 4.7 ± 0.81.5 ± 0.3 1.0 1.4 G1S-A70V 2.9 ± 0.8 6.3 ± 0.5 2.2 ± 0.6 1.5 1.0 T4A 1.8± 0.6 5.5 ± 0.1 3.1 ± 1.0 2.1 1.1 V11A 3.1 ± 0.5 20.9 ± 2.8  6.7 ± 1.34.5 — F16A 1.1 ± 0.4 11.4 ± 2.7  10.3 ± 4.7  6.9 1.8 F25A 1.5 ± 0.5 11.8± 0.1  7.7 ± 0.3 5.1 4.9 R36A 4.0 ± 0.1 4.7 ± 0.2 1.2 ± 0.1 0.8 — P39A2.7 ± 0.2 6.0 ± 0.3 2.2 ± 0.2 1.5 — F49A 2.7 ± 0.7 17.1 ± 0.9  6.3 ± 1.74.2 3.6

[0307] Role of the N-terminal IGF-1 Residues

[0308] Surprisingly, the IGFBP-3 interaction was generally much lessaffected by the alanine substitutions than was the interaction withIGFBP-1, despite the fact that IGFBP-3 binds IGF-1 with approximately10-fold higher affinity. Apart from P63A, no alanine mutant exhibiteda >6-fold reduction in IGFBP-3 affinity (FIG. 14; Table XIII).

[0309] It had previously been shown in biosensor experiments thatdes(1-3)-IGF-1 binds IGFBP-3 with 25-fold reduced affinity (Heding etal., supra). This naturally-occurring form of IGF-1 lacks the firstthree N-terminal residues and shows increased mitogenic potency,presumably due to its reduction in IGFBP-binding (Bagley et al.,Biochem. J., 259: 665-671 (1989)). Since none of the first three aminoacid side chains seem to contribute any energy to the binding of IGFBP-3(Table I) but nevertheless des(1-3)-IGF-1 is compromised in IGFBP-3binding, without being limited to any one theory, it is hypothesizedthat backbone interactions might be involved.

[0310] This hypothesis was tested by displaying on phage a triplealanine mutant (Ala(1-3)- IGF-1), substituting the first threeN-terminal amino acids. If the backbone in that region contributes tothe interaction with IGFBP-3 this mutant should be able to bind. Bindingto IGFBP-1, however, should be reduced due to the lack of the E3 sidechain (Table I). As a control the des(1-2)-IGF-1 mutant was generated,testing for any potential backbone interactions with IGFBP-1 atpositions 1 and 2. As expected, Ala(1-3)-IGF-1 showed a decreasedIGFBP-1 affinity similar to E3A but no change in IGFBP-3 affinity (FIG.14; Table XIII). For des(1-2)-IGF-1, no difference in affinity wasobserved for both binding proteins. Combined with the observations ondes(1-3)-IGF-1 (Heding et al., supra), these results suggest, withoutlimitation to any one theory, that the peptide backbone between residue3 and 4 of IGF-1 mediates important interactions with IGFBP-3.

[0311] Discussion:

[0312] The functional IGFBP-1 and IGFBP-3 binding epitopes on thesurface of IGF-1 have been probed by alanine-scanning mutagenesis. Bothbinding epitopes are illustrated in FIG. 18. Individual IGF-1 side-chaininteractions play a much more important role for binding to IGFBP-1 thanto IGFBP-3. Two major binding patches are found for IGFBP-1 (FIG. 18A).One is situated on the upper face of the N-terminal helix (composed ofG7, L10, V11, L14, F25, 143, and V44) and one the lower face (composedof E3, T4, L5, F16, V17, and L54). These two binding patches are bridgedby F49 and R50. For IGFBP-3, the binding epitope is more diffuse and hasshifted to include G22, F23, and Y24 (FIG. 18B). Binding of IGFBP-3 isgenerally much less sensitive to alanine substitutions. In fact, thebiggest reduction in affinity (apart from P63A, see below) is a 6-folddecrease seen for G7A. This result is intriguing since IGFBP-3 bindswith 10-fold higher affinity to IGF-1 than does IGFBP-1. Most probably,without limitation to any one theory, interactions originating from theIGF-1 main chain backbone are contributing to the binding of IGFBP-3.This hypothesis is further substantiated by the experiments with theAla(1-3)-IGF mutant. While the single and triple alanine substitutionshave no effect on IGFBP-3 binding, deletion of the first three aminoacids resulted in a 25-fold decrease in affinity (Bagley et al., supra;Clemmons et al., Endocrinology, 131: 890-895 (1992); Heding et al.,supra). In summary, IGF-1 uses different binding modes to associate withIGFBP-1 and IGFBP-3: a few amino acid side-chain interactions areimportant for binding to IGFBP-1, while backbone interactions seem toplay a major energetic role for binding to IGFBP-3.

[0313] A recent publication has investigated the binding epitope onIGF-1 for IGFBP-1 by heteronuclear NMR spectroscopy (Jansson et al., J.Biol. Chem., 273: 24701-24707 (1998)). The authors found that the IGF-1residues 29, 30, 36, 37, 40, 41, 63, 65, and 66 amongst othersexperienced chemical shift perturbations upon complexation with IGFBP-1at 30° C. Furthermore, Jansson and co-workers identified R36, R37, andR50 to be part of the functional binding epitope and tested thosealanine mutants in BIACORE™ experiments. The largest change in affinityobserved by these authors was a 3-fold decrease for R50A. However, dueto the structural flexibility of IGF-1 already observed in the first NMRstudy of the hormone (Cooke et al., supra), Jansson et al. were unableto completely assign many residues in the NMR spectrum, including F49.

[0314] In similar studies of protein-protein interfaces it was foundthat only a few side-chain residues contribute to the bulk offree-binding energy (Clackson and Wells, Science, 267: 383-386 (1995);Kelley et al., Biochemistry, 34: 10383-10392 (1995)). The same holdstrue for the IGF-1 GFBP-1 interaction. However, here, as it was noticedfor tissue factor binding to factor VIIa, the magnitude of the freeenergy of binding (ΔΔG) values derived from important side chains issmaller than in the case of growth hormone (Kelley et al., supra). Theresidues with predominant ΔΔG contributions were not clustered on theIGF-1 surface like in the growth hormone-receptor interface (Clacksonand Wells, supra), but still formed a continuous IGFBP-1 binding epitope(FIG. 18A). In contrast, the IGFBP-3 binding epitope on IGF-1 wasdiscontinuous, and side chains contributed very modest individualbinding energies.

[0315] Substitution of P63 by alanine in IGF-1 results in a decreasedaffinity for both binding proteins that cannot be measured in theconcentration range used in the competition phage ELISA's. However,residue P63 is located on the opposite side of the IGF-1 molecule withrespect to the main binding epitope. Furthermore, it has been noticedthat alanine substitutions of glycines and prolines can lead tostructural changes (Di Cera, supra). In addition, Jansson et al., 1998,supra, concluded that the C-terminal part of IGF-1 is not involved indirect IGFBP-1 contacts, but rather undergoes indirect conformationalchanges upon complex formation. An extensive characterization ofantibody binding sites on IGF-1 has been carried out by Manes et al.,Endocrinology, 138: 905-915 (1997). They showed simultaneous binding ofIGFBP-1 or -3 to IGF-1 in complex with antibodies recognizing theC-terminal D-domain. These results further support earlier observationsthat the D-domain, beginning with residue P63, is not involved inbinding of IGFBP-1 or -3 (Bayne et al., supra, 1988).

[0316] The major discrepancy between an IC₅₀ ratio obtained by phageELISA and a BIACORE™ result was observed with residue F16. As alreadymentioned substitution of this residue by alanine induced structuralchanges in the IGF-1 molecule (Jansson et al., supra, 1997). The sameeffect was seen with the K_(D) in the BIACORE™ results, but the affinitydecrease was less pronounced in the phage ELISA experiments (see TableII). Both BIACORE™ measurements used IGF-F16A that had been refoldedduring the purification procedure (Jansson et al., supra, 1997). Inphage display, however, the protein of interest is translocatednaturally by the secretion machinery of E. coli. The low proteinabundance in monovalent phage display (<1 molecule per phage particle)may disfavor aggregation and misfolding. Additionally, fusing IGF-1 tothe truncated g3 phage protein might exert a stabilizing effect on thenative structure of the peptide.

[0317] The levels of IGFBP-3 are positively regulated by IGF-1. The roleof IGFBP-1, in contrast, is less clear. This class of binding proteinsis generally less abundant than IGFBP-3, and its levels are negativelyregulated by insulin (Bach and Rechler, supra; Clemmons, supra, 1997;Jones and Clemmons, supra).

[0318] Based on the results herein, IGFBP-specific variants of IGF-1 areobtained. Combination of several alanine mutations generates a variantthat binds IGFBP-1 very weakly while retaining high-affinity binding ofIGFBP-3. The design of IGFBP-1 specific variants that no longer bind toIGFBP-3, can involve phage display of IGF-1 and the randomization ofamino acids at specific positions (Cunningham et al., 1994, supra;Lowman and Wells, J. Mol. Biol., 234: 564-578 (1993)).

[0319] Conclusion:

[0320] Residues in IGF-1 important for binding to IGFBP-1 and IGFBP-3have been identified. Several residues were found that determine thebinding specificity for a particular IGFBP. Recent publications (Loddicket al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al.,supra, 1998) have reported animal studies where increased pools ofbioavailable “free” IGF-1 were generated by displacing endogenous IGF-1from binding proteins. IGFBP-specific IGF-1 variants may be useddiagnostically and therapeutically as described herein.

Example 7 Characterization of Certain IGF-1 Analogs

[0321] Materials and Methods:

[0322] Construction of IGF-1 Analogs

[0323] In Example 6 (and in Dubaquié and Lowman, supra) IGF-1 analogsare identified in which binding affinity to IGFBP-1, IGFBP-3, or bothbinding proteins, was reduced. In particular, the total alanine-scanningmutagenesis of IGF-1 identified glutamic acid 3 (E3) and phenylalanine49 (F49), as well as phenylalanine 16 (F16) and phenylalanine 25 (F25)to some degree, as specificity determinants for binding to IGFBP-1.Phage display alanine-scanning results suggested that both of the sidechains at positions 3 and 49 selectively contribute considerable bindingenergy for complex formation with IGFBP-1 (˜30-fold loss in affinity forE3A, ˜100 fold for F49A), while their contribution in binding energy forIGFBP-3 is not detectable (E3A) or minor (˜4-fold for F49A) (see Example1 and Dubaquié and Lowman, supra).

[0324] Further improved specificity for IGFBP-3 was likely to beattained by cumulative mutation of IGF-1, because the effects of pointmutations are often additive with respect to their contribution to thefree energy of binding (Wells, Biochemistry 29: 8509 (1990)). Therefore,a double mutant of IGF-1, E3A/F49A, was constructed by combining pointmutations E3A and F49A in a single molecule. Although F16A showed asmaller IGFBP-specificity effect (Example 1 and Dubaquié and Lowman,supra), the double mutant F16A/F49A was also constructed.

[0325] Also constructed was a new point mutant of IGF-1, Y31C,containing a single putative unpaired cysteinyl thiol, to facilitatesite-specific immobilization of IGF-1 for binding assays. Y31C waschosen because it is outside the binding epitopes for IGFBP-1 andIGFBP-3 (Dubaquié and Lowman, supra). This immobilization techniqueensures a uniform ligand population (Cunningham and Wells, J. Mol.Biol., 234: 554 (1993)) for binding by the injected analyte (i.e., IGFbinding protein). The advantage of this method over thepreviously-employed amine coupling is that the IGF-1 N-terminus isunblocked and free of any potential amine linkages to the chip matrix.This may be especially important for binding analysis of IGFBP-1, whichis believed to interact with side chains of the IGF-1 N-terminus(Dubaquié and Lowman, supra). Y31C displayed on phage showedwild-type-like affinities for both IGFBP-1 and IGFBP-3, supporting thenotion that the region around residue 31 is important in receptorbinding, but forms no contact with the binding proteins (Bayne et al.,J. Biol. Chem., 264: 11004 (1988); Bayne et al., J. Biol. Chem., 265:15648 (1990)).

[0326] Single-alanine variants of IGF-1, including F49A, as well as theE3A/F49A double mutant, were expressed, purified, and refolded to givethe appropriate disulfide isomer as judged by HPLC analysis (Example 1herein and Dubaquié and Lowman, supra). These variants were tested inassays of specific binding-protein binding and receptor activation.

[0327] Results:

[0328] IGFBP-1 and IGFBP-3 Binding Affinity

[0329] The binding affinities of these variants for IGFBP-1 and IGFBP-3were compared to that of wild-type IGF-1 using BIACORE™ analysis.Kinetic experiments with IGFBP-3 binding to immobilized IGF-1 orvariants (Table III) were carried out as described in Example 1 and inDubaquié and Lowman, supra, and compared with F49A IGF-1 and wild-typeIGF-1. In this assay, the double mutant E3A/F49A was about 20-foldweaker in binding affinity to IGFBP-3 than wild-type, and the doublemutant F16A/F49A was about 66-fold weaker (Table XV). TABLE XV Kineticsof IGFBP-3 Binding to IGF-1 (*data from Table II of Example 1)Immobilized IGFBP-3 Protein k_(a) (×10⁵ M⁻¹) k_(d) (×10⁻⁴ s⁻¹) K_(D)(nM) IGF-1* 3.2 ± 0.5 4.7 ± 0.8 1.5 ± 0.3 F49A IGF-1* 2.7 ± 0.7 17.1 ±0.9  6.3 ± 1.7 E3A/F49A IGF-1 0.74 ± 0.4  13.3 ± 0.6  22.2 ± 10.3F16A/F49A IGF-1 0.4 ± 0.1 38.6 ± 2.7  99.0 ± 26.0

[0330] For measurements of IGFBP-1 binding to IGF-1, kineticsexperiments were conducted using a single-cysteine IGF-1 variant, Y31C,that was immobilized onto the sensor chip surface via a disulfidelinkage (BIACORE™ System Manual Supplement, 5a-1, Pharmacia (1991)). Theresults are consistent (Table XVI) with the binding affinity measuredusing wild-type IGF-1 immobilized via nonspecific amine coupling to thebiosensor chip (Example 1 and Dubaquié and Lowman, supra). TABLE XVIKinetics of IGFBP-1 Binding to IGF-1 (*data from Table XIV of Example 6)Immobilized IGFBP-1 Protein k_(a) (×10⁴ M⁻¹) k_(d) (×10⁻⁴ s⁻¹) K_(D)(nM) Y31C IGF-1 3.9 ± 0.4 3.8 ± 0.1 10.0 ± 1.1 IGF-1* 3.2 ± 0.2 4.1 ±0.2 13.0 ± 1.0

[0331] The binding of F49A and E3A/F49A to IGFBP-1 was too weak foraccurate kinetic measurements. Therefore, a competitive binding assay(WO 98/45427 published Oct. 15, 1998) was performed to estimate thecorresponding affinities. The single-cysteine IGF-1 variant, Y31C, wasused that was immobilized onto a BIACORE™ biosensor chip surface asdescribed above. Competitive binding experiments yielding half-maximalinhibitory concentration values (IC₅₀) were conducted as follows: 50 nMIGFBP-1 was incubated with a dilution series of the desired IGF variant.These protein mixture solutions were injected at 5 μL/min over a B1 chipcontaining cysteine-coupled IGF-1 Y31C (200 response units). The amountof bound IGFBP-1 was determined by subtracting non-specific bindingafter a 20-minute injection and plotted against the IGF variantconcentration (FIG. 19). The results are shown in Table XVII. TABLE XVIIInhibition of IGFBP-1 Binding to Immobilized Y31C IGF-1 IGFBP-1Immobilized Protein Competing Protein IC₅₀ (μM) Y31C IGF-1 F49A IGF-11.6 ± 0.2 Y31C IGF-1 E3A/F49A IGF-1 64 ± 9 

[0332] Compared to wild-type IGF-1, F49A and E3A/F49A had severelydecreased binding affinities for IGFBP-1. F49A bound to IGFBP-1 with anIC₅₀ of 1.6 0.2 μM (Table XVII), while preserving a high-affinitydissociation constant (K_(D)) of 6.3+1.7 nM for IGFBP-3 (Table XV).Binding of E3A/F49A to IGFBP-1 was found to be even weaker, with anestimated IC₅₀ of 64+9 μM (Table XVII), while having only moderatelyreduced affinity (K_(D)=22.2±10.3 nM) for IGFBP-3 (Table XV). These invitro measurements suggest that neither IGF variant should stablyassociate with IGFBP-1 under physiological conditions.

[0333] KIRA Assays of IGF Type I Receptor Activation

[0334] The Kinase Receptor Activation Assay (KIRA) specifically andquantitatively monitors the extent of cytoplasmic IGF receptorphosphorylation upon extracellular stimulation by ligand (Sadick et al.,J. Pharm. Biomed. Analysis, 19 (6): 883-891 (1999)). Several IGFvariants, G15/A70V, T4A, V11A, F16A, F25A, F16A/F49A, R36A, P39A, andF49A, were tested in single-concentration assays of receptor activation.The IGFBP-1 and IGFBP-3 binding affinities of these variants, except forF16A/F49A, are set forth in Table XIV and in Dubaquié and Lowman, supra.Table XVIII summarizes the relative affinities and specificities fromBIACORE™ measurements.

[0335] For the KIRA assay, variant concentrations were roughly estimatedat 13 nM (“high concentration) or 1.3 nM (“low concentration”), based onoptical density measurements. The signal obtained for each IGF variantwas compared to that of a standard-dilution series of wild-type IGF-1,and reported in terms of an apparent IGF-1 concentration correspondingto the observed activity in the KIRA assay (FIGS. 20A and 20B). Althoughexact relative potencies were not measured, these results show that alltested mutants maintain the ability to activate the IGF type I receptor.TABLE XVIII Relative IGFBP-1 and IGFBP-3 Affinities of IGF-1 Variants.NDB, no detectable binding; ND, not determined; *data from Table XIV ofExample 6) IGFBP-1 IGFBP-3 Specificity IGF-1 K_(D) (mutant) / K_(D)(mutant) / Relative BP-1 / Variant K_(D) (IGF-1)) K_(D) (IGF-1))Relative BP-3 G1S/A70V* 1.1 1.5 0.7 T4A* 6.9 2.1 3.3 V11A* 5.1 4.5 1.1F16A* 25 6.9 3.6 F25A* 25 5.1 4.9 R36A* 1.1 0.8 1.4 P39A* 1.0 1.5 0.7F49A* 70 4.2 16.7 F16A/F49A ND 65.6 ND

[0336] Table XVIII shows that, in addition to F49A, F16A and F25A areboth substantially reduced in affinity for IGFBP-1, but less so forIGFBP-3. Both still retain biological activity based on KIRA assays(FIG. 20).

[0337] For determining relative potency of F49A and E3A/F49A, theirability to activate the type I IGF receptor was measured using serialdilutions in KIRA assays. As shown in FIGS. 21A-21B, both F49A andE3A/F49A display IGF receptor activation curves that areindistinguishable from wild-type IGF-1. The half-maximal effectiveconcentrations (EC₅₀) were 20.0 ±1.3 ng/ml for F49A, 19.8±0.5 ng/ml forE3A/F49A, and 18.9±0.2 ng/ml and 19.8±0.6 ng/ml for wild-type IGF-1.These results strongly suggest that both IGF mutants are fullybiologically active.

[0338] Blood Clearance and Renal Accumulation of IGF-1 Variants in Rats

[0339] To assess preliminary pharmacological properties of F49A andE3A/F49A IGF-1, both proteins were radiolabeled and administeredintravenously to rats. FIG. 22A shows a time course of the rate at whichboth molecules are cleared from the blood of the animals. As expecteddue to their decreased IGFBP affinities, both variants were cleared at afaster rate compared to wild-type human IGF-1. Interestingly, the doublemutant (E3A/F49A) was cleared faster than the single mutant (F49A),correlating well with the respective affinities for the major bindingprotein in the serum, IGFBP-3 (Table XV). FIG. 22B shows thetissue-to-blood ratio for the IGF variants in different organs. Themajority of the radioactively-labeled IGF molecules were detected in thekidney, whereas radioactivity levels in the liver, spleen, heart, andpancreas were much lower. It is evident that the variants F49A andE3A/F49A accumulate at statistically significant higher levels in thekidney compared to wild-type IGF-1.

[0340] Circular Dichroism Analysis of IGF-1 Variants

[0341] The circular dichroism spectra of F49A and E3A/F49A IGF-1 wereanalyzed to test whether the introduced mutations cause major changes tothe protein structure. Structural destabilization could lead toincreased proteolytic susceptibility, providing an alternativeexplanation for the faster blood clearance rates of the IGF variants. Asshown in FIG. 23, however, both mutants have virtually identical spectrato the one recorded for wild-type IGF-1. The CD spectra reveal elementsof both α-helix and random coil, as expected from NMR spectroscopy ofIGF-1 (Cooke et al., supra). The thermal stability of IGF-1 could not bedetermined accurately by circular dichroism, presumably due to therelatively high content (˜30%) of random coil (Jansson et al., supra,1997) already present at room temperature. The fact that the CD spectraof both variants showed no significant deviation from wild-type IGF-1 isan indication that the introduced mutations do not alter the overallstructure of IGF-1.

[0342] Conclusion:

[0343] From the evidence presented above, it would be expected that thesingle and double mutants F16A, F16G, F16S, F25A, F25G, F25S, F49A,F49G, F49S, E3A/F49A, E3A/F49G, E3G/F49A, E3G/F49G, E3A/F49S, E3S/F49A,E3S/F49S, E3G/F49S, and E3S/F49G IGF-1 would be effective in treatingcartilage disorders, since the alanine-substituted mutants exhibit areduced affinity for IGFBP-1 without substantial loss of ability to bindto IGFBP-3 and are biologically active based on many tests. Further,such mutants are expected to be efficacious in treating cartilagedisorders, since the alanine-substituted mutants only weakly bind toIGFBP-1 and there is disregulation in IGFBP-3 present in arthriticdisorders (Martel-Pelletier et al., supra). It would also be expectedthat BP3-01 and BP3-15 would also be efficacious for this purpose inview of their role in displacing IGFBP-3 (Lowman et al., supra, 1998; WO98/45427).

Example 8 Articular Cartilage Explant Assay of IGF-1 Analogs WithSelectively Reduced Affinity for IGFBP-1 versus IGFBP-3

[0344] Introduction:

[0345] The experiments of this example examine both the synthetic andprophylactic potential of the IGF-1 analogs E3A/F49A and F49A on thecartilage matrix.

[0346] Materials and Methods:

[0347] Articular Cartilage Explants

[0348] The metacarpophalangeal joint of 18-24 month old cows wasaseptically dissected, and articular cartilage was removed by free-handslicing, taking care so as to avoid the underlying bone. The cartilagewas minced, washed, and cultured in bulk for at least 24 hours in ahumidified atmosphere of 95% air and 5% CO₂ in serum-free (SF) LGDMEM/F12 medium with 0.1% BSA, 100 U/ml penicillin/streptomycin (Gibco),2 mM L-Glutamine, 0.1 mM MEM sodium pyruvate (Gibco), 20 μg/mlGENTAMICIN™ (Gibco) antibiotic, and 1.25 mg/L AMPHOTERICIN B™ (Sigma)antibiotic. Articular cartilage was aliquoted into MICRONICS™ tubes(approximately 55 mg per tube) and incubated for at least 24 hours inthe above media. Control (media alone), wild-type IGF-1, E3A/F49A, orF49A was then added to each tube (to a final concentration of 40 or 400ng/ml as indicated). The media was harvested and changed at various timepoints (0, 24, 48 and 72 hours).

[0349] Proteoglycan Release

[0350] Medium harvested at various time points was assayed forproteoglycan content using the 1,9-dimethylmethylene blue (DMMB)colorimetric assay of Farndale and Buttle, Biochem. Bhiophys. Acta, 883:173-177 (1986). A standard curve was prepared of chondroitin sulfateranging from 0.0 to 5.0 μg.

[0351] Proteoglycan Synthesis

[0352] After the media change at 48 hours, ³⁵S-sulfate (to a finalconcentration of 10 μCi/ml) was added to the cartilage explant cultures.After an additional 17 hours of incubation at 37° C., the amount ofproteoglycans in the media was measured using the DMMB assay. Thecartilage pieces themselves were washed twice with explant media anddigested overnight at 50° C. in a 900 μL reaction volume of 10 mM EDTA,0.1 M sodium phosphate, and 1 mg/ml proteinase K (Gibco BRL). 600 μl ofthe digestion reaction was mixed (2:1) with 10% w/v cetylpyridiniumchloride (Sigma) and centrifuged at 1000×g for 15 minutes. Thesupernatant was removed and formic acid (500 μl, Sigma) was added todissolve the pellets.

[0353] The samples were then transferred to scintillation vialscontaining 10 ml scintillation fluid (ICN) and read in a scintillationcounter.

[0354] Remaining Proteoglycan in Cartilage Tissues

[0355] After 72 hours, the remaining articular cartilage explants weredigested as described above under Proteoglycan synthesis and assayed forproteoglycan content using the DMMB colorimetric assay (referenced aboveunder Proteoglycan release).

[0356] Nitric Oxide Assay

[0357] Articular cartilage media, saved from the cartilage explants atvarious times (24, 48, and 72 hours), was mixed with 10 μl 0.05 mg/ml2,3-diaminonapthalene (DAN) in 0.62M HCl and incubated at roomtemperature for 10-20 minutes in the dark. The reaction was terminatedwith 5 μl of 2.8N NaOH. The amount of fluorescence of2,3-diamionaphthotriazole was measured with a CYTOFLOR™ fluorescentplate reader at 365-nm excitation at 409-nm emission.

[0358] Results:

[0359] The data presented in FIGS. 24-27 show that the two IGF-1 analogstested decrease cartilage matrix breakdown (as measured by proteoglycanrelease), block the induction of cartilage matrix breakdown by IL-1α,induce cartilage matrix synthesis (as measured by proteoglycansynthesis), and prevent inhibition of matrix synthesis by IL-1α.

[0360] These effects are similar to those of wild-type IGF-1. However,since human IGF-1 is employed with bovine tissue, the speciesdifferences may mask an inhibitory effect of endogenous binding proteinson wild-type IGF-1 activity.

[0361] The IGF-1 analogs significantly decreased nitric oxide release(FIG. 28). In addition, the IGF-1 analogs blocked induction of nitricoxide by IL-1α (FIG. 29).

[0362] Discussion:

[0363] It is shown herein that IGF-1 analogs are capable of inhibitingmatrix breakdown, stimulating new matrix synthesis, and inhibitingnitric oxide release. In addition, these IGF-1 analogs inhibit thedetrimental effects of interleukin 1, which is elevated in diseasedjoints.

[0364] The role of nitric oxide in breakdown of articular cartilage,especially the destruction associated with osteoarthritis has beendescribed in Amin et al., Curr. Opin. Rheum., 10: 263-268 (1998). Sincenitric oxide also has effects on other cells, the presence of nitricoxide within the joint could increase vasodilation and permeability,potentiate cytokine release by leukocytes, and stimulate angiogenicactivity by monocyte-macrophages. Normal cartilage does not producenitric oxide unless stimulated with cytokines such as IL-1, whileosteoarthritic cartilage explants continue to release nitric oxide forover 3 days in culture despite the absence of added stimuli. Thus,production of nitric oxide by cartilage correlates with a diseasedstate, and since nitric oxide appears to play a role in both the erosiveand the inflammatory components of joint diseases, a protein or peptidethat decreases nitric oxide production would likely be beneficial forthe treatment of degenerative cartilagenous disorders. In fact, in vivoanimal models suggest that inhibition of nitric oxide production reducesprogression of arthritis (Pelletier et al., Arthritis Rheum., 41(7):1275-1286 (1998); van de Loo et al., Arthritis Rheum., 41: 634-646(1998); Stichtenoth and Frolich, Br. J. Rheumatol., 37: 246-257 (1998)).

[0365] The assay described herein is based on the principle that2,3-diaminonapthalene (DAN) reacts with nitrite under acidic conditionsto form 1-(H)-naphthotriazole, a fluorescent product. As nitric oxide isquickly metabolized into nitrite (NO₂ ⁻¹) and nitrate (NO₃ ⁻¹),detection of nitrite is one means of detecting (albeit undercounting)the actual nitric oxide produced in cartilagenous tissue.

[0366] As described above, nitric oxide has detrimental effects onchondrocytes as well as other cell types within the joint. Sinceinhibition of nitric oxide has been shown to inhibit progression ofarthritis in animals, the effect of the IGF analogs on nitric oxidefurther suggests that the tested IGF analogs would be protective forjoint tissues in vivo. Finally, these analogs or the IGFBP displacerpeptides are expected to have anabolic effects on tissues, such asarthritic cartilage, which are otherwise IGF-1 resistant.

[0367] In summary, two IGFBP-selective variants (F49A and E3AF49A)demonstrated a 700-fold and 80,000-fold apparent reduction in affinityfor IGFBP-1, while preserving low nanomolar affinity for IGFBP-3, themajor carrier of IGF-1 in plasma. Both variants displayed wild-type-likepotency in cellular receptor kinase assays, stimulated human cartilagematrix synthesis, and retained their ability to associate with ALS incomplex with IGFBP-3. Hence, the half-life of these variants is stilldetermined by IGFBP-3, but their activity is no longer regulated byIGFBP-1. Furthermore, pharmacokinetic parameters and tissue distributionof these two IGF-1 variants in rats differed from wild-type IGF-1 as afunction of their IGFBP affinities.

Example 9 Generation of IGF-1 Analogs with Selectively Reduced Affinityfor IGFBP-3 Versus IGFBP-1 and Articular Cartilage Explant AssayTherefor

[0368] Introduction:

[0369] The IGFBPs are generally thought to inhibit the biologicalactivity of IGF-1 by sequestering the growth factor into high-affinitycomplexes and thereby preventing its receptor association (Jones andClemmons, Endocr. Rev. 16: 3-34 (1995)). The levels of IGFBP-3 (andIGFBP-4) were found to be increased in human inflammatory synovial fluid(Kanety et al., J. Rheumatol. 23: 815-818 (1996)). This change in IGFBPhomeostasis is thought to contribute to the pathological condition bydepriving cells from the IGF-1 survival signal. In this example IGF-1molecules were generated with selectively reduced affinity for IGFBP-3,without altering activity on the IGF type I receptor. Such moleculeswould be predicted to be more biologically potent than wild-type IGF-1in the presence of elevated pathophysiological IGFBP-3 levels.Furthermore, since IGFBP-3 is the major carrier of IGF-1 in serum, thehalf-life and biological distribution of such IGF-1 variants wouldpresumably be drastically altered.

[0370] The binding epitopes of IGFBP-1 and IGFBP-3 have been mapped byalanine-scanning on the surface of IGF-1 (Dubaquié and Lowman,Biochemistry, 38: 6386-6396 (1999)). Each individual IGF-1 side-chaincontributes only modest amounts of binding energy for IGFBP-3. Based onthis observation it seems impossible to substantially decrease IGFBP-3affinity by introducing only a few specific alanine mutations intoIGF-1. A different strategy to disrupt protein-protein interactionsinvolves the introduction of a charged residue into the bindinginterface, leading to electrostatic repulsion of the binding partners.It has been noted by Jansson et al. (Biochemistry, 36: 4108-4117 (1997))that IGF-1 is an electrostatically polarized protein with a continuousnegatively-charged patch at the N-terminus (including the B-regionhelix), while the C-region is mainly positively charged. Based on theseobservations residue D12 was selected, since it does not contribute anybinding energy for IGFBP-1 and seems to be part of the structuralIGFBP-3 binding epitope (Dubaquié and Lowman, supra). It was reasonedthat replacing residue 12 with a positive charge would disrupt thecontinuous negatively-charged patch that might possibly be involved inthe IGFBP-3 interaction.

[0371] Materials and Methods:

[0372] Generation of Analogs

[0373] Phage particles displaying IGF-1 variants in which the aspartateresidue at position 12 was mutated to lysine (D12K) or arginine (D12R)were constructed as described in Dubaquié and Lowman, supra.Furthermore, these protein variants were expressed in E. coli andpurified as described in Dubaquié and Lowman, supra.

[0374] Articular Cartilage Explants

[0375] The metacarpophalangeal joint of a six-month old pig was culturedas described above for bovine articular cartilage in Example 8. Thisexplant was cultured in media alone or in media with D12K, D12R, orwild-type IGF-1 (at 10 nM) alone or in the presence of IL-1α at 1 ng/ml,as described above. Human explants were from patients undergoing jointreplacement surgery. Articular cartilage was harvested, aliquoted (40-45mg/tube) and cultured as above. Twenty-four hours later, explants weretreated with 40 ng/ml IGF-1, F16A/F49A, E3A/F49A, or F49A, every day forfive days. Fresh media was added on days 0, 1, 2, and 3. Matrixbreakdown was determined by measuring the amount of proteoglycans in themedia using the DMMB assay as set forth above. Matrix (proteoglycan)synthesis was determined by measuring ³⁵S-sulfate uptake as set forthabove.

[0376] Further, for comparison purposes, F49A, E3A/F49A, F16A/F49A,D12K, D12R, or wild-type IGF-1 (at 40 ng/ml) were tested for cartilagematrix synthesis in human tissue as described above. Human articularcartilage from diseased joints was cultured in media alone or with F49A,E3A/F49A, F16A/F49A, D12K, D12R or wild-type IGF-1 (at 40 ng/ml) andmatrix synthesis was determined by measuring ³⁵S-sulfate uptake asdescribed above.

[0377] Results:

[0378]FIG. 30 shows the binding curves of phage particles displayingeither wild-type IGF-1, D12K, or D12R bound to immobilized IGFBP-1 (FIG.30A) or IGFBP-3 (FIG. 30B). The wild-type IGF-1 phage particles bound toIGFBP-1 or IGFBP-3 generated similar detection signal intensities. D12Kand D12R, however, displayed lower signal intensities when binding toIGFBP-3 compared to IGFBP-1. This result suggests that these variantshave a selectively-reduced affinity for IGFBP-3 compared to wild-typeIGF-1.

[0379] Like IGF-1, D12K and D12R inhibited cartilage matrix breakdown(FIG. 31A) and increased matrix synthesis (FIG. 31C). In addition, likeIGF-1, D12K or D12R inhibited the catabolic effects of IL-1α0 (FIG. 31B)and prevented IL-1α-induced inhibition of proteoglycan synthesis (FIG.31D). Thus, these mutants retain full activity, and are expected to begood therapeutic agents for cartilage disorders such as arthritis, whichare characterized by increased matrix breakdown and decreased matrixsynthesis. In addition, since high levels of IL-1 are found in diseasedjoints, the ability of these mutants to inhibit the detrimental effectsof IL-1α on cartilage further suggest utility of these IGF variants astreatments for arthritis.

[0380] Importantly, the IGF-1 variants have activity on human cartilageobtained from patients undergoing joint replacement, i.e., witharthritis. As shown in FIG. 32, all five IGF variants tested withselective preferences for IGFBP-3 over IGFBP-1 or vice-versa (F49A,E3AF49A, F16A/F49A, D12K, and D12R) stimulated matrix synthesis indiseased human articular cartilage, and the activity of the variants wasas good as, or better than, that of wild-type IGF-1. Unlike previousstudies showing IGF-1 resistance in articular cartilage fromosteoarthritic joints, cartilage from these particular patients remainedresponsive to IGF-1.

[0381] Discussion:

[0382] The protein interface between IGF-1 and IGFBP-3 seems to besensitive to mutations changing the charge distribution. Introducingpositive charges into the N-terminal region of IGF-1 is expected toselectively reduce IGFBP-3 affinity.

[0383] The IGF-1 variants D12K and D12R are biologically active, asshown in the articular matrix synthesis stimulation experiments herein.

[0384] IGF-1 is a key regulator of matrix homeostasis in articularcartilage. The metabolic imbalance in osteoarthritis that favors matrixbreakdown over new matrix synthesis may be due, at least in part, toinsensitivity of chondrocytes to IGF-1 stimulation. While the mechanismunderlying this IGF-1 resistance is not known, without being limited toany one theory, it is believed that IGFBPs, which are elevated in manyarthritic patients, play a role. In these patients, IGF-1 analogs thatdo not bind to, and are thus not inhibited by, IGFBPs would likelystimulate cartilage repair in tissue that is otherwise IGF-1 resistant.Alternatively, peptides that block IGF-1 binding to inhibitory bindingproteins may thus free IGF-1 to act on resident chondrocytes.Furthermore, based on the possible role of IGFBPs (especially IGFBP-1)in modifying the activity of IGF-1, the IGF-1 analogs described hereinmay have better clearance from, and/or transport through tissues within,human joints relative to that of wild-type IGF-1.

[0385] Since loss of cartilage matrix proteins is an early andcontinuous part of joint damage leading to joint failure, the ability ofthese IGF-1 analogs to inhibit cartilage catabolism and stimulate newmatrix synthesis strongly suggests that these IGF analogs will havebeneficial effects on diseased or damaged joints.

[0386] Il-1αhas catabolic effects on cartilage, including the generationof synovial inflammation, up-regulation of matrix metalloproteinases,stimulation of matrix breakdown, and inhibition of proteoglycan andcollagen synthesis. Furthermore, IL-1 protein is found in diseased, butnot normal joints. Thus, the ability of the tested analogs to havepositive effects on cartilage, as well as to counteract the deleteriouseffects of IL-1α, strongly suggests that such molecules would have aprotective effect on cartilage disorders, including damaged and/ordiseased cartilage. In addition, such an activity predicts that the testIGF-1 analogs and peptides would inhibit the degradation that occurs inarthritic conditions, since antagonism of IL-1α function has been shownto reduce the progression of osteoarthritis (Arend et al., Ann. Rev.Immunol., 16: 27-55 (1998)).

Example 10 Articular Cartilage Explant Assay of BP3-15 and BP3-40Analogs

[0387] Materials and Methods:

[0388] Human articular cartilage explants were cultured in media ortreated with IGF-1 by itself or in combination with either BP3-40 orBP3-15 at 0.1 mg/ml as set forth above. Matrix breakdown was determinedby measuring the release of proteoglycans into media as described above.Matrix synthesis was determined by counting the amount of ³⁵S-sulfateincorporated into the cartilage tissue as described above.

[0389] Results and Discussion:

[0390] Since high levels of IGFBPs have been found in diseasedcartilage, and these IGFBPs may inhibit IGF-1 activity, the effect oftwo IGFBP-3 displacer peptides on IGF-1 activity was tested. Humanarticular cartilage explants from arthritic patients were treated withIGF-1 alone or in combination with an IGFBP displacer peptide (BP3-15 orBP3-40). These displacer peptides appeared to enhance the ability ofIGF-1 to decrease matrix breakdown (FIGS. 33A, 33C) and to stimulatematrix synthesis (FIGS. 33B, 33D). Thus, these peptides are expected tobe useful for conditions, such as arthritis, where high levels ofinhibitory binding proteins are present. In such conditions, treatmentwith a displacer peptide alone, or in combination with IGF-1, isexpected to enhance the anabolic effects of endogenous or exogenousIGF-1 and to be useful in treating arthritis.

Example 11 Complex Formation With ALS

[0391] Materials and Methods:

[0392] Human ALS was expressed in CHO cells (Leong et al., Mol.Endocrinol., 6: 870-876 (1992)). The secreted ALS was enriched onDEAE-Sepharose, followed by affinity purification on an IGF-1/IGFBP-3column, as described in Baxter et al., J. Biol. Chem., 264: 11843-11848(1989).

[0393] To monitor trimeric complex formation, 300 RU's of biotinylatedIGFBP-3 (Sulfo-NHS-LC-LC-biotin; Pierce, Rockford, Ill.) wereimmobilized on a streptavidin-coupled chip (SA; Biacore, Inc.,Piscataway, N.J.) in PBS, 0.05% TWEEN™-20, and 0.01% sodium azide. Thebiosensor chip was primed with running buffer containing 1 μM of therespective IGF-1 analog (F49A or E3A/F49A). ALS was injected in amountsof 98, 148, and 333 nM at 50 μL/min. for 2 minutes, followed by a2-minute dissociation period. The chip was regenerated with 10 μL of 2MKSCN (in 50 mM HEPES pH 7.2) at a flow rate of 20 μL/min., followed by a3-minute buffer flow for baseline stabilization.

[0394] Results:

[0395] F49A and E3A/F49A Form Ternary Complexes With IGFBP-3 and ALS

[0396] The majority of IGF-1 in serum is found in a 150-kDa ternarycomplex composed of IGFBP-3 and a glycoprotein termed ALS. The half-lifeof this ternary complex is an order of magnitude longer than that offree IGF-1(Ferry et al., Horm. Metab. Res., 31: 192-202 (1999)). Hence,ternary complex formation is a major determinant for IGF-1biodistribution. To test whether the engineered IGF-1 variants werestill able to form trimeric complexes with ALS, a biosensor bindingexperiment was performed. Biotinylated IGFBP-3 was immobilized on abiosensor chip, and ALS was injected having either wild-type IGF-1 (FIG.34A), F49A (FIG. 34B), or E3A/F49A (FIG. 34C) included in the runningbuffer at a concentration of 1 μM. As shown in FIG. 34, the ALS bindingcurves were essentially identical for the wild-type IGF-1 and the twoIGF-1 variants. The dissociation rate for wild-type IGF-1 and each IGF-1variant is listed in Table XIX, ranging from 4.0×10⁻⁴s⁻¹ to 6.3×10⁻⁴s⁻¹.In the absence of any IGF-1 in the running buffer, ALS did not associatewith IGFBP-3 on the chip. This experiment indicates that the IGF-1variants retain the ability to form ternary complexes, and that theirterminal half-life in serum therefore should not be drastically shorterthan for wild-type IGF-1. TABLE XIX ALS Dissociation Rates fromIGFBP-3/IGF-1 complexes determined by Biosensor Experiments MaterialDissociation Rate kd (×10⁻⁴s⁻¹) wt IGF-1 6.3 ± 1.7 F49A IGF-1 4.2 ± 0.9E3A/F49A IGF-1 4.0 ± 2.2

[0397] Clearance and Distribution of F49A and E3A/F49A in Rats.

[0398] Human wild-type IGF-1, F49A, and E3A/F49A were radiolabeled andadministered intravenously to rats to assess their pharmacokineticproperties. Both IGF-1 variants cleared significantly faster whencompared to wild-type human IGF-1 (Table XX). E3A/F49A cleared at thefastest rate (1107 ±45 ml/hr/kg), followed by F49A (427±125 ml/hr/kg)and wt IGF-1 (151 ±24.7 ml/hr/kg). A similar trend was observed for theintermediate half-life t_(1/2) ^(β): E3A/F49A had the shortest, F49Aintermediate, and wt IGF-1 the longest t_(1/2) ^(β) (Table XX). Thesefindings correlate well with the corresponding in vitro affinities forthe major binding protein in the serum, IGFBP-3. Interestingly, thesteady-state distribution volumes were much greater for the IGF-1variants, indicating that they are less confined to the plasmacompartment than is wild-type IGF-1 (Table XX). The greatesttissue-to-blood ratios for the IGF-1 molecules were detected in thekidney, whereas ratios in the liver, spleen, heart, and pancreas weremuch lower. It is evident from this experiment that F49A and E3A/F49Aaccumulate at statistically significant higher ratios in the kidneycompared to wild-type IGF-1. TABLE XX Pharmacokinetic parametersfollowing a single IV dose in rats. steady- state distribution Half-life(hr) clearance volume Group t_(1/2) ^(α) t_(1/2) ^(β) t_(1/2) ^(γ)(ml/hr/kg) (ml/kg) ¹²⁵I-wt IGF-1 0.033 ± 0.004 1.22 ± 0.237  5.88 ±0.374 151 ± 24.7 936 ± 94.8 ¹²⁵I-F49A 0.064 ± 0.052 0.971 ± 0.336⁺  8.97± 4.50   427 ± 125*⁺  3201 ± 488*⁺ ¹²⁵I-E3A/F49A 0.032 ± 0.005 0.321 ±0.091* 6.23 ± 1.47 1107 ± 45.0* 6520 ± 874* 

[0399] Discussion:

[0400] The ability of the analogs F49A and E3A/F49A to form ternarycomplexes with ALS and IGFBP-3 has important consequences for the serumhalf-life of these variants. How ALS interacts with the IGF-1/IGFBP-3complex is unknown due to a lack of structural information on IGFBP-3and ALS. A recent model of ALS postulates a donut-shaped structure whoseinternal cavity is lined with negative charges; these regions couldpotentially interact with positive charges on IGFBP-3 and IGFBP-5(Janosi et al., J. Biol. Chem., 274: 5292-5298 (1999)). The affinity ofALS for a crosslinked IGF-1/IGFBP-3 complex is on the order of 0.1 to0.3 nM, depending on the carbohydrate content of ALS (Janosi et al.,supra). In the biosensor measurements that analyzed ALS binding to anon-covalent IGF-1/IGFBP-3 complex (by including saturating amounts ofIGF-1 in the running buffer), the dissociation rates of the wild-typeIGF-1, F49A, and E3A/F49A remained constant between 4.0×10⁻⁴s⁻¹ to6.3×10⁻⁴s⁻¹ (Table XIX). The experiment confirms that both IGF-1variants tested form ternary complexes with ALS.

[0401] The pharmacokinetics of the IGF-1 variants in rats showeddifferent characteristics from wild-type IGF-1 (Table XX). It appearsthat the majority of differences are observed in the first 60-100minutes of the experiment. In the pharmacokinetic analysis model, thisphase is characterized by the initial and intermediate half-livest_(1/2) ^(α) and t_(1/2) ^(β), which likely describe the distribution offree and total radiolabeled molecules to extravascular tissues and theirclearance (Table XX). Following this initial phase, all three IGF-1molecules are cleared at comparable rates, as suggested by their similarterminal half-life values t_(1/2) ^(γ), which likely reflect theelimination of ¹²⁵-I from the rat (Table XX). The trends in t_(1/2) ^(α)and t_(1/2) ^(β) observed across the three molecules are generallyconsistent with a more rapid rate of distribution and clearance for theIGF-1 variants that are less bound to IGFBPs. Furthermore, thecalculated steady-state distribution volumes and clearance rates alsosupport the interpretation that IGFBP association plays a major role inthe distribution and clearance of the IGF-1 variants. Therefore, withoutbeing limited to any one theory, this suggests that the initialdifferences in the pharmacokinetic profile reflect the IGF-1/IGFBP-3binding equilibrium, since they correlate well with the correspondingaffinities of the IGF-1 variants for IGFBP-3 in vitro.

[0402] Because IGFBP-3 in the blood seems to be saturated under normalconditions (Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)), it isbelieved, without limitation to any one theory, that the injected IGF-1variants have to compete with endogenous IGF-1 for IGFBP-3 binding. TheIGF-1 molecules that are unable to associate with IGFBP-3 could diffuseout of the vasculature, and either associate with other binding proteinsor be cleared at a rapid rate (t_(1/2) for free IGF-1=10-15 min¹¹).Those molecules that achieve ternary complex formation with IGFBP-3 andALS, however, experience a more prolonged serum half-life and become“trapped” in the vasculature. Ternary complex formation of the IGF-1variants occurs with the same efficiency as wild-type IGF-1 in vitro(FIG. 33, Table XIX), and this property appears to be reflected in thelater phase (>100 minutes) of the pharmacokinetic profile. The fact thatboth human IGF-1 variants and wild-type human IGF-1 display terminalhalf-lives in the order of 6-9 hours strongly suggests that complexformation with rat IGFBP-3 and ALS occurs in vivo. This is important,since all in vitro experiments were done exclusively with human IGFBP-3and ALS, whereas the in vivo experiments rely on preservation of theengineered specificities for the homologous rat binding proteins.

Example 12 Substitutions in BP1-16

[0403] Several single-residue substitutions in BP1-16 were tested fortheir effect on IGFBP-1 binding affinity by synthesizing peptides andmeasuring inhibition of IGFBP-1 binding to IGF-1. Sites for substitutionwere chosen based upon the known effect of an alanine or othersubstituted residue at the site.

[0404] G4 was previously found to be substitutable by D-alanine. Becausethe conformational effects of D-alanine are different from those ofL-alanine, L-alanine was substituted for G4 in peptide BP1-29.Inhibition assays showed a 50-fold loss in binding affinity with thissubstitution (Table XXI).

[0405] P5 was previously found to be highly conserved in phage-displayedpeptide libraries; however, some substitutions were observed. Forexample, three different peptide-phage clones were found with arginineat this position. Therefore, the L-alanine substitution for proline wastested, as well as several alternative substitutions (BP1-30, BP1-31,BP1-34). The results (Table XXI) show that P5A, P5N, and P5R are welltolerated.

[0406] L6 and L9 were completely conserved in 40 of 40 sequenced clonesand 61 of 61 sequenced clones, respectively, from two different IGFBP-1selected peptide-phage libraries. In addition, substitution of either ofthese residues with L-alanine or aib (alpha-aminoisobutyrate) side-hainsresulted in a significant loss in IGFBP-1 binding affinity. Two furthersubstitutions were tested at each position: norleucine (Nle), an isomerof leucine, or arginine (the aliphatic portion of the side-chain ofwhich might still be able to pack into the peptide structure). While theArg substitutions resulted in peptides having undetectable IGFBP-1affinity (BP1-32 and BP1-26), the Nle substitutions were well-tolerated(BP1-36 and BP1-37). The non-natural substitutions L6(Nle) and L9(Nle)are therefore the only substitutions at these positions known topreserve moderate-affinity binding to IGFBP-1.

[0407] W8 was also completely conserved in IGFBP-1 selectedpeptide-phage libraries, although the alanine substitution had a smallereffect on binding than in the case of L6 or L9. Therefore, several largeside-chain substitutions were tested at this position. Interestingly,arginine, 1-naphthylalanine (Nal(l)), or histidine substitutions(BP1-22, BP1-23, and BP1-24, respectively) each had modest (<10-fold)effects on IGFBP-1 binding affinity (Table XXI). From these experiments,a new consensus sequence for IGFBP-1 binding may be formulated asfollows:

[0408]CysXaa₍₆₎Xaa₍₇₎GlyXaa₍₉₎LeuXaa₍₁₁₎TrpLeuCysXaa₍₁₅₎Xaa₍₁₆₎Xaa₍₁₇₎Xaa₍₁₈₎(SEQ ID NO:130), where Xaa₍₆₎, Xaa₍₇₎, Xaa₍₉₎, Xaa₍₁₁₎, Xaa₍₁₅₎, andXaa₍₁₆₎ are independently any amino acid, and Xaa₍₁₂₎, Xaa₍₁₇₎, andXaa₍₁₈₎ are independently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met.TABLE XXI Relative affinities of BP1-16 variants measured by ELISA orBIAcore ™(*) inhibition assays Fold potency reduction BP1-16 PeptideIC₅₀ (mut) / Variant Sequence IC₅₀ (BP1-16) BP1-16 CRAGPLQWLCEKYF (SEQID NO:35) -1- BP1-29 CRAAPLQWLCEKYF (SEQ ID NO:131) 50 BP1-30CRAGALQWLCEKYF (SEQ ID NO:132) 1.5 BP1-31 CRAGRLQWLCEKYF (SEQ ID NO:133)2.0 BP1-34 CRAGNLQWLCEKYF (SEQ ID NO:134) 3.1 BP1-32 CRAGPRQWLCEKYF (SEQID NO:135) >1000 BP1-36 CRAGPLLQWLCEKYF (SEQ ID NO:136), 6.9 where theunderlined L is Nle BP1-26 CRAGPLQWRCEKYF (SEQ ID NO:137) >570 BP1-37CRAGPLQWLCEKYF (SEQ ID NO:138), 1.7 where the underlined L is Nle BP1-22CRAGPLQRLCEKYF (SEQ ID NO:139) 3.3* BP1-23 CRAGPLQALCEKYF (SEQ IDNO:140), 4.8* where the underlined A is Nal (1) BP1-24 CRAGPLQHLCEKYF(SEQ ID NO:141) 7.5

Example 13 Minimization of the BP1-01 Peptide via “Locked Helix”

[0409] It was previously shown that removal of the disulfide bond inBP1-01 is destabilizing to both structure and function of the peptide.The possibility has been investigated of replacing the disulfide bond ofBP1-01 with a chemically distinct structural constraint, whilemaintaining moderate binding affinity to IGFBP-1. These constraints weredesigned to link side-chain positions separated by 7 (from position i toposition i+7) or 8 (from i to i+8) residues in the BP1-01 peptide.

[0410] The i+7 locked helix strategy, one of the approaches used herein,has been described by Phelan et al., J. Am. Chem. Soc., 119: 455-460(1997); WO 98/20036 published May 14, 1998, as have other i+7, i+3, andi+4 linkages (reviewed in Phelan et al., supra). In addition, otherside-chain substitutions, allowing for ionic or hydrophobic interactionsor metal chelation, have been used for the purpose of stabilizing ahelical structure (reviewed by Phelan et al., supra). Herein isdescribed a novel i+8 locked helix strategy, which is particularlyuseful for stabilization of the helical structure found in the BP1-01peptide family.

[0411] Mutagenesis studies indicated that major determinants for IGFBP-1binding reside primarily in the helical segment of BP1-01. Theseimportant binding determinants segregate mainly to one face of thehelix, and include Leu6 and Leu9, and the aromatic residues Trp8, Tyr13,and Phe14. Without being limited to any one theory, the remainder of thepeptide might act primarily to stabilize the helix and to ensureappropriate presentation of the major side-chain binding determinants.Therefore, other methods for constraining the binding segment of thepeptide to a helical conformation might yield potent BP1-bindingpeptides. Side-chain-side-chain crosslinks on the opposite helical facefrom the major BP1-binding determinants were chosen for use. This methodhas been described in WO 98/20036, supra. In the present case, the i+7crosslinking connects residues replacing Gln7 to Phe14, which replacesone of the hydrophobic IGFBP-1 binding determinants.

[0412] The i+8 crosslinking connects residues replacing Gln7 to Gly15.

[0413] The crosslinking chemistry involves replacement of theappropriate two residues with glutamic acid residues (the first and lastGlu (E) residues shown in Table XXII), where the two Glu residues arejoined by forming amides with 1,5-diaminopentane. This cross-linkingmethod has been described in WO 98/20036, supra.

[0414] To develop active peptides shorter than BP1-01, it was alsodecided to delete the disulfide (Cys1-Cys10) and truncate the N-terminalloop region in constrained helical peptides. The Cys10 was changed toAla and Cys1 replaced with an acetyl group (ac in Table XXII). Severalshorter variants lacked one or more of the other loop residues. Thus,these peptides were cyclized only through the 1,5-diaminopentanelinkage. Such peptides, lacking disulfide bonds, may be more stable todegradation in vitro and in vivo. They may also be reduced inimmunogenicity compared to disulfide-containing analogs.

[0415] Functional Analysis of Locked Helices

[0416] Peptides were assayed in a BIAcore™ assay as described in WO98/45427, supra. These inhibition assays (FIG. 35) compared the relativepotency of these peptides for blocking the interaction of IGFBP-1 withIGF-1. Adding the “i+7 helical lock” to a variant of BP1-01 reducedrelative potency (Table XXII) by 6-fold (peptide (i+8)C) to 8-fold(peptide (i+7)D or (i+8)B) in the best locked-helix variants. Thesepeptides demonstrate that a disulfide bond is not necessary to obtainstructured, functional peptides of the BP1-01 family.

[0417] In contrast to the locked helix variants described above, alocked helix variant in which two of the key IGFBP-1 bindingdeterminants were lost ((i+7)A; Table XXII) exhibited significant lossin binding activity relative to BP1-01. In this peptide, W8 is replacedwith the first cross-linking residue and Gly15 is replaced with thesecond cross-linking residue; F14 is replaced by alanine in thispeptide. The disulfide bond is still present in this peptide.

[0418] Certain additions to the N-terminus and C-terminus of thesepeptides (see Example 3) are predicted to improve their binding affinityand potency, as in the case of disulfide-constrained peptide variantsdiscussed below.

[0419] Hence, a consensus sequence can be formulated as follows:

[0420] Xaa₍₁₋₄₎Xaa₍₅₎Xaa₍₆₋₇₎ProLeuGluXaa₍₁₁₎LeuAlaXaa₍₁₄₎Xaa₍₁₅₎Xaa₍₁₆₎Xaa₍₁₇₎GluXaa₍₁₉₎ (SEQ ID NO:142), wherein Xaa₍₁₋₄₎ is absent or isbetween 1 and 4 amino acids of any kind; Xaa₍₅₎ is any amino acid,Xaa₍₆₋₇₎ is absent or is between 1 and 2 amino acids, Xaa₍₁₄₎ andXaa₍₁₅₎ are independently any amino acid, Xaa₍₁₁₎ and Xaa₍₁₆₎ areindependently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met, Xaa₍₁₇₎ isabsent or is 1-napthyl-Ala, His, Phe, Trp, Tyr, Pro, Gln, or Met, andXaa₍₁₉₎ is absent or is Gly.

[0421] NMR Analysis of Locked Helices

[0422]¹H NMR spectroscopy was used to ascertain that the locked helixvariants of BP1-01 did have the desired three-dimensional helicalstructure. 1-dimensional spectra and 2-dimensional COSY, TOCSY, andROESY spectra were acquired for peptides (i+7)A, (i+7)B, (i+7)C, (i+7)Dand (i+8)C; experimental details were similar to those described forBP1-01 in Lowman et al., supra, 1998. Preliminary analysis of backbone³JHN-Ha scalar coupling constants (derived from 2D COSY spectra) andshort Ha(i)-HN (i+3) distances (derived from ROESY spectra), indicatedthat for (i+7)A, (i+7)C, (i+7)D, and (i+8)C, the designed helix waspresent. In the case of (i+7)B, the NMR data were not consistent with ahelical structure. The lack of a well-folded structure presumablyexplains the low affinity of this peptide for IGFBP-1 (>360 fold weakerthan BP1-01)

[0423] The scalar coupling and ROESY data for (i+7)A, (i+7)D, and (i+8)Cwere analyzed in more detail to generate input restraints for thecalculation of three-dimensional structures as described previously forBP1-01 (Lowman et al., supra, 1998). Comparison of the minimized meanstructures of the locked helix variants to that of BP1-01 yielded RMSDs(N,Ca,C atoms of Leu6-Phe14) of 1.02 A and 0.22 A for (i+7)D and (i+8)C,respectively. Further, the packing of hydrophobic side-chains Leu6,Trp8, Leu9, and Tyr13 in these two locked helix variants was also verysimilar to the packing in BP1-01. Thus, the (i,i+7) and (i,i+8) lockedhelix scaffolds have successfully maintained many aspects of the BP1-01structure without the need for a disulfide bond. Although the covalenttethers in (i+7)A did produce the desired two turns of helix (the N,Ca,CRMSD between minimized means of BP1-01 and (i+7)A is 1.06 A), someside-chain rotamers differed significantly from those of BP1-01.

[0424] The structural analyses described above suggest that covalenttethers (other than the disulfide bond observed in BP1-01) may be usedto control peptide structure. The use of i,i+7 or i,i+8 tethers producedpeptides (i+7)D and (i+8)C that retained high affinity towards IGFBP-1in the absence of a disulfide bond. Presumably, the affinity derivesfrom stabilization of a structure that maintains both the backbonehelical fold and the side-chain packing arrangement of the key bindingdeterminants observed in BP1-01. Although the peptide (i+7)A maintainsthe backbone fold, two of the key determinants (Trp8 and Phe14) aremissing, and the orientation of others (e.g. Tyr13) is perturbed; as aresult, this peptide has reduced affinity. The peptide (i+7)B fails toadopt the desired fold, and hence has no measurable affinity forIGFBP-1. TABLE XXII Locked-helix variants of BP1-01 (The first and lastGlus (Es) are sites of cyclizing “lock”) Fold potency reduction BP1-16Peptide IC₅₀ (BP1-01) / Variant Sequence IC₅₀ (mut) BP1-01CRAGPLQWLCEKYFG (SEQ ID NO:26) -1- (i + 7)A acCRAGPLQELCEKYAE (SEQ IDNO:143) 40 (i + 7)B acLEWLAEKYEG (SEQ ID NO:144) >360 (i + 7)CacPLEWLAEKYEG (SEQ ID NO:145) 20 (i + 7)D acRAGPLEWLAEKYEG (SEQ IDNO:52) 7.7 (i + 8)A acLEWLAEKYFE (SEQ ID NO:146) >200 (i + 8)BacRPLEWLAEKYFE (SEQ ID NO:53) 7.7 (i + 8)C acRAGPLEWLAEKYFE (SEQ IDNO:54) 5.9

Example 14 N-Terminal Variants of BP1-16

[0425] Previous affinity-maturation experiments led to a peptideaddition to 10 the C-terminus of BP1-02, including a number ofpeptide-phage clones (Table XXIII), and the synthetic peptide BP1-21A,the sequence of which is shown in Table XXIII. Table XXIII illustratesthe C-terminal substitutions in the background of BP1-02. Table XXIIIC-terminal substitutions derived from round 3 of monovalent phageselections in the BP1-02 peptide background BP1-02 Peptide Number ofclones Variant Sequence SEQ ID NO: sequenced Y135C (BP1-21A)SEVGCRAGPLQWLCEKYFSTY 13 2 Y135D SEVGCRAGPLQWLCEKYFATY 14 3 Y135FSEVGCRAGPLQWLCEKYFQTY 15 1 Y135B SEVGCRAGPLQWLCEKYFQTYT 16 1 Y135ASEVGCRAGPLQWLCEKYFDTY 17 1 Y135E SEVGCRAGPLQWLCEKYFETY 18 1 Y135KSEVGCRAGPLQWLCEKYFKTY 19 1

[0426] It is sought herein to improve affinity further by two methods:substitution of the first four N-terminal amino acid residues fromBP1-20 into BP1-21A, and re-randomization of the N-terminal amino acidresidues of BP1-21A (in the context of the previously improvedC-terminus).

[0427] Peptide BP1-25 (Table XXV) was synthesized to test the additivity(Wells, Biochemistry, 29: 8509-8517 (1990)) for the N-terminal andC-terminal maximally-preferred substitutions. Compared with BP1-16 ininhibition assays, BP1-25 showed about a 20-fold affinity improvement.However, the affinity of BP1-25 was not significantly improved overBP1-21A. This affinity improvement was confirmed in other assaysdescribed below.

[0428] In the second approach, a monovalent-display peptide-phagelibrary, presenting BP1-21A as a fusion to g3p, was randomized (Lowman,Methods Mol. Biol., 87: 249-264 (1998)) at the N-terminal four residues.Binding selection to IGFBP-1 was carried out by first allowing libraryphage to bind to solution biotinylated IGFBP-1, with an initialconcentration of 50 nM, followed by 28 nM for the subsequent four roundsof selection. Peptide-phage capable of binding IGFBP-1 were captured byincubating with streptavidin magnetic beads (Promega) for 10 minutes atroom temperature. For each round of selection, the washing was graduallymodified to be more stringent. Off-rate selection was performed byadding 2.5-5 μM IGF in solution to prevent rebinding of phage withfaster off-rates. It is of interest to note that for the last round ofselection (round 5), with an overnight incubation at 4° C. in thepresence of 2.5 μM IGF, there were still phage remaining bound to thebeads (2.2×10⁴ total phage were eluted). Subsequent sequencing datarevealed that 14 out of 20 selected clones had converged to a single DNAsequence (clone Y0791A; Table XXIV). A peptide corresponding to thissequence, BP1-40, was synthetically produced for analysis. TABLE XXIVN-terminal substitutions derived from round 5 of monovalent phageselections in the BP1-21A peptide background Number BP1-16 Peptide SEQID of clones Variant Sequence NO: sequenced Y0791A GQQSCRAGPLQWLCEKYFSTY21 14  (BP1-40) Y0791D ASSMCRAGPLQWLCEKYFSTY 22 1 Y0791HQGPDCRAGPLQWLCEKYFSTY 23 1 Y0791K QASECRAGPLQWLCEKYFSTY 24 1 Y0791LAETLCRAGPLQWLCEKYFSTY 25 1 Y0791S NSLLCRAGPLQWLCEKYESTY 26 1 Y0791TAQWVCRAGPLQWLCEKYFSTY 27 1

[0429] Inhibition assays for measuring relative potencies of peptidesfor inhibiting IGFBP-1 binding to IGF-1 have been described (e.g., WO98/45427, supra). Peptides described herein were of sufficient bindingaffinity to allow for direct measurement of binding affinities bysurface plasmon resonance (SPR) using a BIAcore™ system. The directbinding kinetics of IGFBP-1 peptides were measured by injecting a seriesof 2-fold diluted peptides in running buffer (0.05% TWEEN 20™ in PBS)over a carboxy-methyl (CM) biosensor chip coupled with about 590-1000 RUof IGFBP-1 at a flow rate of 50 μl/min on a BIAcore-2000 ™ orBIAcore-3000™ instrument. The immobilization of IGFBP-1 was performedthrough EDC/NHS chemistry as described by the manufacturer. Peptideswere also injected through a flow cell containing IGFBP-3 as backgroundcontrol. Since the off-rate for most of the peptides is relatively fast(in the range of 2×10⁻² s⁻¹), off-rate measurement was set for 30minutes. This allowed for regeneration of IGFBP-1 on the chip by simpledissociation, rather than by addition of eluent. For each dilution ofpeptides, a global fit of the sensorgram data was performed using a 1:1Langmuir binding model. On-rates ranged from 4×10⁵ to 1.9×10⁶ M⁻¹ s⁻¹ .The binding affinities, K_(D), calculated as k_(off)/k_(on) aresummarized in Table XXV. Peptides BP1-20, BP1-21A, BP1-25, and BP1-40were all found to have similar binding affinities (K_(D)) of about 20 nMto 40 nM.

[0430] The conclusion from these experiments is that N-terminalextensions to the BP1-01 peptide can improve binding affinity (as inBP1-02, BP1-20, BP1-21A, BP1-25, BP1-40, and other variants identifiedin Table XXIV). Some substitutions may alter expression levels in E.coli, since GQQS (SEQ ID NO:147) was clearly selected fromphage-displayed peptide libraries. However, peptides having thesequences SEVG (SEQ ID NO:148), SEMV (SEQ ID NO:149), EARV (SEQ IDNO:150), or GQQS (SEQ ID NO:151) at their N-termini all had similarbinding affinities. Therefore, the nature of added side-chains at theN-terminus appears to have little effect upon peptide binding affinity.This suggests that main-chain interaction of the peptide in this regionmay contribute to binding affinity for IGFBP-1. An improved consensussequence for IGFBP-1 binding peptides is expected therefore to be:

[0431]Xaa₍₁₋₄₎CysXaa₍₆₎Xaa₍₇₎GlyXaa₍₉₎LeuXaa₍₁₅₎Xaa₍₁₂₎LeuCysXaa₍₁₅₎Xaa₍₁₆₎Xaa₍₁₇₎Xaa₍₁₈₎(SEQ ID NO:152), wherein Xaa₍₁₄₎ is absent or is between 1 and 4 aminoacids of any kind, Xaa₍₆₎, Xaa₍₇₎, Xaa₍₉₎, Xaa₍₁₁₎, Xaa₍₁₅₎, (andXaa₍₁₆₎ are independently any amino acid, and Xaa₍₁₂₎, Xaa₍₁₇₎, andXaa₍₁₈₎ are independently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met.As noted in Example 1, truncation of the amino-terminal 4 residues(Xaa₍₁₋₄₎) has only a small effect on activity, giving a shorterconsensus that still retains binding:

[0432]CysXaa₍₆₎Xaa₍₇₎GlyXaa₍₉₎LeuXaa₍₁₁₎TrpLeuCysXaa₍₁₅₎Xaa₍₁₆₎Xaa₍₁₇₎Xaa₍₁₈₎(SEQ ID NO:130). TABLE XXV Peptide affinity determinations by BIAcore ™kinetics BP1-16 Peptide K_(D) ± (SD Variant Sequence or SE) (nM) BP1-02SEVGCRAGPLQWLCEKYFG (SEQ ID 210 ± 46  NO: 50) BP1-20 EARVCRAGPLQWLCEKYF(SEQ ID 33 ± 15 NO: 39) BP1-21A SEVGCRAGPLQWLCEKYFSTY (SEQ ID 41 ± 17NO: 40) BP1-25 EARVCRAGPLQWLCEKYFSTY (SEQ ID 42 ± 11 NO: 42) BP1-40GQQSCRAGPLQWLCEKYFSTY (SEQ ID 27 ± 21 NO: 43)

Example 15 Cell-Based Assay of Peptide Activity

[0433] A cell-based (KIRA) assay was previously described for measuringthe amount of IGF-like activity displaced by peptides from mixtures ofIGF-1 and binding proteins (Lowman et al., supra, 1998; WO 98/45427,supra). The KIRA assay was used to compare in vitro bioactivity ofBP1-16, BP1-02, BP1-25, and BP1-40. In this example, very lowconcentrations of IGF-1 and IGFBP-1 were used, i.e., below the K_(D) Ofthe peptide: 2 nM [IGF-1] and 1.5 nM [IGFBP-1], with a titration seriesof [peptide]=0.1 to 200 nM. IGF-1 and peptide were mixed and added tocells expressing IGF receptor for 30 min, then IGFBP-1 was added for anadditional 1 h.

[0434] Increased potency was observed for both peptides BP1-25 andBP1-40 over peptides BP1-16 and BP1-02 (FIG. 36). However, under theseconditions, BP1-02 was not significantly more active than BP1-16; andBP1-40 was not significantly more active than BP1-25. The EC₂₀(concentration at which 20% of maximal IGF-1 activity is observed)values were 10-20 nM for BP1-25 and BP1-40, and 150-200 nM for BP1-16and BP1-02.

Example 16 Biosynthesis of a BP1-01 Peptide Variant

[0435] An additional variant of BP1-21A was designed for peptidebiosynthesis in E. coli. For this approach, a DNA sequence encoding thepeptide was fused by site-directed mutagenesis to the gene for aconsensus domain of protein-A known as Z-domain (Nilsson et al., ProteinEngineering, 1(2):107-113 (1987). After expression and secretion from E.coli, the fusion protein was enzymatically cleaved with trypsin to yieldfree peptide, which can be purified from the enzymatic reaction mix(see, e.g., Varadarajan et al., PNAS, 82:5681-5684 (1985);Castellanos-Serra et al., FEBS, 378:171-176 (1996); Nilsson et al., J.Biotechnology, 48:241-250 (1996).

[0436] A detailed procedure for trypsin digestions has been described inSmith, Methods in Mol. Biol., 32:289-296 (1994). Because this proteaseis highly specific for Arg and Lys residues, the BP1-40 peptide wasmodified by mutation of these residues for construction of the fusion.From previous mutagenesis and phage-library results, it was known thatArg and Lys residues of BP1-01 could be substituted without significantloss of binding affinity. Therefore, a fusion protein was designed withsubstitutions R2A and K12H (numbering is according to the BP1-01sequence).

[0437] Furthermore, BP1-01, having a Gly residue following theC-terminal F14 of BP1-16, was known to have no significant effect onbinding affinity. Therefore, a Gly-Arg sequence was added at the end ofthe peptide to allow for trypsin cleavage. The sequences of theBP1-625-Z fusion protein and the BP1-625 peptide (as cleaved by trypsin)are given in Table XXVI. TABLE XXVI Peptide sequences for E. colibiosynthesis Construct Peptide sequence BP1-625-ZGQQSCAAGPLQWLCEHYFSTYGRGGGSGGAQHDEAVDNKFNKE (SEQ ID NO:47)QQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLN DAQAPNVDMN BP1-625GQQSCAAGPLQWLCEHYFSTYGR (SEQ ID NO:46)

[0438] The fusion protein BP1-625-Z was produced from E. colishake-flask cultures. Culture supernatants were sterile-filtered, thenapplied to an IgG-Sepharose™ column (Pharmacia). The bound fraction waseluted with 1M acetic acid, then lyophilized and resuspended intrypsin-digest buffer: 10 mM Tris (pH 8.0), 100 mM NaCl, 1 mM CaCl₂.TPCK-treated trypsin (Sigma) was added at a weight/weight ratio of 1:100to 1:200 (trypsin to substrate) and digestion was carried out at 25° C.for 1-2 hours. Thereafter, PMSF was added to 1 mM to stop the reaction.Samples were adjusted to 1 mM TFA and run on an analytical HPLC columnwith a 0-60% acetonitrile gradient in 0.1% TFA. The two predominantpeaks were collected (FIG. 37) and shown by mass spectrometry tocorrespond to a Z-domain fragment, and the peptide BP1-625.

[0439] The peptide BP1-625 fraction was lyophilized and resuspended in100 mM HEPES buffer, pH 7.2. Inhibition experiments were carried out ina BIAcore assay as previously described, except that limiting amounts(9-10 nM IGFBP-1) were used to make the assay sensitive with respect toaffinities in the 10⁻⁸ M range. These assays showed that the BP1-625peptide blocked IGFBP-1 binding to immobilized IGF-1 and was similar inactivity to BP1-25, having about 20-fold improved potency over BP1-01(FIG. 38).

[0440] It may be predicted that BP1-625 will block IGF-1 binding toIGFBP-1 and produce IGF-like activity on cells, with similar potency toBP1-21A, BP1-25, or BP1-40. It would also be expected that a peptide,BP1-625T, comprising the sequence:

[0441] GlyGlnGlnSerCysAlaAlaGlyProLeuGlnTrpLeuCysGluHisTyrPheSerThrTyr(SEQ ID NO:153) would act similarly to BP1-625.

[0442] The BP1-625-Z fusion is useful for producing IGFBP-bindingpeptides from E. coli, and the Z part of the fusion can beadvantageously attached to other peptides herein than just BP1-625.

Example 17 Articular Cartilage Explants from Human Joints

[0443] Materials and Methods:

[0444] The same Material and Methods (for human tissue) were used asdescribed above, using articular cartilage explants from human jointsremoved from patients undergoing joint replacement. These explants werecultured with IGF-1 alone at 40 ng/ml, or IGF-1 with BP1-17, BP3-15, orBP1-16 (0.1 mg/ml), or IGF-1 with buffer (HEPES).

[0445] Proteoglycan breakdown and synthesis were measured as describedabove.

[0446] Results and Discussion:

[0447] The role of specific IGFBPs in IGF-1 activity was tested bytreating articular cartilage explants from patients undergoing jointreplacement with IGF-1 in the presence of peptides that inhibit IGF-1binding to particular IGFBPs. In particular, BP1-16 inhibits IGF-1binding to IGFBP-1, and BP3-15 inhibits IGF-1 binding to IGFBP-3. BP1-17binds with much lower affinity to IGFBP-1. In addition, buffer alone(100 mM HEPES) was included as a negative control.

[0448] As shown in FIG. 39, both BP3-15 and BP1-16, and also BP1-17 to alesser extent, enhance the protective effect of IGF-1. Namely, BP3-15and BP1-16, and to some extent BP1-17, enhance anabolism, as shown bythe increase in matrix synthesis. Thus, these three peptides, andespecially BP3-15 and BP1-16, are expected to be useful therapeutics forthe treatment of arthritis.

[0449] The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the objectives of the presentinvention.

1 153 1 5140 DNA Artificial sequence Sequence is synthesized 1gaattcaact tctccatact ttggataagg aaatacagac atgaaaaatc 50 tcattgctgagttgttattt aagcttgccc aaaaagaaga agagtcgaat 100 gaactgtgtg cgcaggtagaagctttggag attatcgtca ctgcaatgct 150 tcgcaatatg gcgcaaaatg accaacagcggttgattgat caggtagagg 200 gggcgctgta cgaggtaaag cccgatgcca gcattcctgacgacgatacg 250 gagctgctgc gcgattacgt aaagaagtta ttgaagcatc ctcgtcagta300 aaaagttaat cttttcaaca gctgtcataa agttgtcacg gccgagactt 350atagtcgctt tgtttttatt ttttaatgta tttgtaacta gtacgcaagt 400 tcacgtaaaaagggtatcta gaggttgagg tgattttatg aaaaagaata 450 tcgcatttct tcttgcatctatgttcgttt tttctattgc tacaaatgcc 500 tatgcatctg gtaccgccat ggctgatccgaaccgtttcc gcggtaaaga 550 tctggcaggt tcaccaggtg gaggatccgg aggaggcgccgagggtgacg 600 atcccgcaaa agcggccttt aactccctgc aagcctcagc gaccgaatat650 atcggttatg cgtgggcgat ggttgttgtc attgtcggcg caactatcgg 700tatcaagctg tttaagaaat tcacctcgaa agcaagctga taaaccgata 750 caattaaaggctccttttgg agcctttttt tttggagatt ttcaacgtga 800 aaaaattatt attcgcaattcctttagttg ttcctttcta ttctcactcc 850 gctgaaactg ttgaaagttg tttagcaaaaccccatacag aaaattcatt 900 tactaacgtc tggaaagacg acaaaacttt agatcgttacgctaactatg 950 agggttgtct gtggaatgct acaggcgttg tagtttgtac tggtgacgaa1000 actcagtgtc tagctagagt ggcggtggct ctggttccgg tgattttgat 1050tatgaaaaga tggcaaacgc taataagggg gctatgaccg aaaatgccga 1100 tgaaaacgcgctacagtctg acgctaaagg caaacttgat tctgtcgcta 1150 ctgattacgg tgctgctatcgatggtttca ttggtgacgt ttccggcctt 1200 gctaatggta atggtgctac tggtgattttgctggctcta attcccaaat 1250 ggctcaagtc ggtgacggtg ataattcacc tttaatgaataatttccgtc 1300 aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt1350 agcgctggta aaccatatga attttctatt gattgtgaca aaataaactt 1400attccgtggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg 1450 tattttctacgtttgctaac atactgcgta ataaggagtc ttaatcatgc 1500 cagttctttt ggctagcgccgccctatacc ttgtctgcct ccccgcgttg 1550 cgtcgcggtg catggagccg ggccacctcgacctgaatgg aagccggcgg 1600 cacctcgcta acggattcac cactccaaga attggagccaatcaattctt 1650 gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg1700 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg 1750tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct 1800 aggctggcggggttgcctta ctggttagca gaatgaatca ccgatacgcg 1850 agcgaacgtg aagcgactgctgctgcaaaa cgtctgcgac ctgagcaaca 1900 acatgaatgg tcttcggttt ccgtgtttcgtaaagtctgg aaacgcggaa 1950 gtcagcgccc tgcaccatta tgttccggat ctgcatcgcaggatgctgct 2000 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga2050 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt 2100accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta 2150 tcgtgagcatcctctctcgt ttcatcggta tcattacccc catgaacaga 2200 aattccccct tacacggaggcatcaagtga ccaaacagga aaaaaccgcc 2250 cttaacatgg cccgctttat cagaagccagacattaacgc ttctggagaa 2300 actcaacgag ctggacgcgg atgaacaggc agacatctgtgaatcgcttc 2350 acgaccacgc tgatgagctt taccgcagga tccggaaatt gtaaacgtta2400 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 2450aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 2500 cgagatagggttgagtgttg ttccagtttg gaacaagagt ccactattaa 2550 agaacgtgga ctccaacgtcaaagggcgaa aaaccgtcta tcagggctat 2600 ggcccactac gtgaaccatc accctaatcaagttttttgg ggtcgaggtg 2650 ccgtaaagca ctaaatcgga accctaaagg gagcccccgatttagagctt 2700 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa2750 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 2800caccacaccc gccgcgctta atgcgccgct acagggcgcg tccggatcct 2850 gcctcgcgcgtttcggtgat gacggtgaaa acctctgaca catgcagctc 2900 ccggagacgg tcacagcttgtctgtaagcg gatgccggga gcagacaagc 2950 ccgtcagggc gcgtcagcgg gtgttggcgggtgtcggggc gcagccatga 3000 cccagtcacg tagcgatagc ggagtgtata ctggcttaactatgcggcat 3050 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac3100 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 3150cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 3200 actcaaaggcggtaatacgg ttatccacag aatcagggga taacgcagga 3250 aagaacatgt gagcaaaaggccagcaaaag gccaggaacc gtaaaaaggc 3300 cgcgttgctg gcgtttttcc ataggctccgcccccctgac gagcatcaca 3350 aaaatcgacg ctcaagtcag aggtggcgaa acccgacaggactataaaga 3400 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac3450 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 3500cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3550 cgctccaagctgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3600 cgccttatcc ggtaactatcgtcttgagtc caacccggta agacacgact 3650 tatcgccact ggcagcagcc actggtaacaggattagcag agcgaggtat 3700 gtaggcggtg ctacagagtt cttgaagtgg tggcctaactacggctacac 3750 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg3800 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 3850ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 3900 tcaagaagatcctttgatct tttctacggg gtctgacgct cagtggaacg 3950 aaaactcacg ttaagggattttggtcatga gattatcaaa aaggatcttc 4000 acctagatcc ttttaaatta aaaatgaagttttaaatcaa tctaaagtat 4050 atatgagtaa acttggtctg acagttacca atgcttaatcagtgaggcac 4100 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc4150 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 4200tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4250 taaaccagccagccggaagg gccgagcgca gaagtggtcc tgcaacttta 4300 tccgcctcca tccagtctattaattgttgc cgggaagcta gagtaagtag 4350 ttcgccagtt aatagtttgc gcaacgttgttgccattgct gcaggcatcg 4400 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctccggttcccaa 4450 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag4500 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 4550cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 4600 gtaagatgcttttctgtgac tggtgagtac tcaaccaagt cattctgaga 4650 atagtgtatg cggcgaccgagttgctcttg cccggcgtca acacgggata 4700 ataccgcgcc acatagcaga actttaaaagtgctcatcat tggaaaacgt 4750 tcttcggggc gaaaactctc aaggatctta ccgctgttgagatccagttc 4800 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca4850 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4900ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 4950 atattattgaagcatttatc agggttattg tctcatgagc ggatacatat 5000 ttgaatgtat ttagaaaaataaacaaatag gggttccgcg cacatttccc 5050 cgaaaagtgc cacctgacgt ctaagaaaccattattatca tgacattaac 5100 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa5140 2 77 PRT Artificial sequence Sequence is synthesized 2 Ser Gly ThrAla Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp 1 5 10 15 Leu Ala GlySer Pro Gly Gly Gly Ser Gly Gly Gly Ala Glu Gly 20 25 30 Asp Asp Pro AlaLys Ala Ala Phe Asn Ser Leu Gln Ala Ser Ala 35 40 45 Thr Glu Tyr Ile GlyTyr Ala Trp Ala Met Val Val Val Ile Val 50 55 60 Gly Ala Thr Ile Gly IleLys Leu Phe Lys Lys Phe Thr Ser Lys 65 70 75 Ala Ser 3 70 PRT Homosapiens 3 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln 15 10 15 Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly 2025 30 Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp 35 4045 Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 50 55 60Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala 65 70 4 86 PRT Homo sapiens 4Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu 1 5 10 15Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 ArgArg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly 35 40 45 Gly GlyPro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly 50 55 60 Ser Leu GlnLys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile 65 70 75 Cys Ser Leu TyrGln Leu Glu Asn Tyr Cys Asn 80 85 5 51 PRT Homo sapiens 5 Phe Val AsnGln His Leu Cys Gly Ser His Leu Val Glu Ala Leu 1 5 10 15 Tyr Leu ValCys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 Gly Ile Val GluGln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 35 40 45 Leu Glu Asn Tyr CysAsn 50 6 20 PRT Artificial sequence Sequence is synthesized 6 Ala SerGlu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys 1 5 10 15 Asn MetTrp Gly Arg 20 7 20 PRT Artificial sequence Sequence is synthesized 7Val Ala Trp Glu Val Cys Trp Asp Arg His Asp Gln Gly Tyr Ile 1 5 10 15Cys Thr Thr Asp Ser 20 8 18 PRT Artificial sequence Sequence issynthesized 8 Ala Trp Glu Val Cys Trp Asp Arg His Gln Gly Tyr Ile CysThr 1 5 10 15 Thr Asp Ser 9 18 PRT Artificial sequence Sequence issynthesized 9 Glu Glu Ser Glu Cys Phe Glu Gly Pro Gly Tyr Val Ile CysGly 1 5 10 15 Leu Val Gly 10 18 PRT Artificial sequence Sequence issynthesized 10 Ser Glu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu CysAsn 1 5 10 15 Met Trp Gly 11 18 PRT Artificial sequence Sequence issynthesized 11 Asp Met Gly Val Cys Ala Asp Gly Pro Trp Met Tyr Val CysGlu 1 5 10 15 Trp Thr Glu 12 18 PRT Artificial sequence Sequence issynthesized 12 Thr Gly Val Asp Cys Gln Cys Gly Pro Val His Cys Val CysMet 1 5 10 15 Asp Trp Ala 13 18 PRT Artificial sequence Sequence issynthesized 13 Thr Val Ala Asn Cys Asp Cys Tyr Met Pro Leu Cys Leu CysTyr 1 5 10 15 Asp Ser Asp 14 15 PRT Artificial sequence Sequence issynthesized 14 Ser Glu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu CysAsn 1 5 10 15 15 15 PRT Artificial sequence Sequence is synthesized 15Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn Met Trp Gly 1 5 10 15 1612 PRT Artificial sequence Sequence is synthesized 16 Val Cys Trp ProVal Ala Glu Trp Tyr Leu Cys Asn 1 5 10 17 11 PRT Artificial sequenceSequence is synthesized 17 Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 15 10 18 13 PRT Artificial sequence Sequence is synthesized 18 Glu ValCys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 19 14 PRT Artificialsequence Sequence is synthesized 19 Glu Glu Val Cys Trp Pro Val Ala GluTrp Tyr Leu Cys Asn 1 5 10 20 16 PRT Artificial sequence Sequence issynthesized 20 Ala Ser Glu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr LeuCys 1 5 10 15 Asn 21 15 PRT Artificial sequence Sequence is synthesized21 Ser Glu Glu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 1522 15 PRT Artificial sequence Sequence is synthesized 22 Ser Glu Glu ValCys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 15 23 15 PRTArtificial sequence Sequence is synthesized 23 Gly Pro Glu Thr Cys TrpPro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 15 24 12 PRT Artificialsequence Sequence is synthesized 24 Cys Gln Leu Val Arg Pro Asp Leu LeuLeu Cys Gln 1 5 10 25 11 PRT Artificial sequence Sequence is synthesized25 Ile Pro Val Ser Pro Asp Trp Phe Val Cys Gln 1 5 10 26 15 PRTArtificial sequence Sequence is synthesized 26 Cys Arg Ala Gly Pro LeuGln Trp Leu Cys Glu Lys Tyr Phe Gly 1 5 10 15 27 19 PRT Artificialsequence Sequence is synthesized 27 Ser Glu Val Gly Cys Arg Ala Gly ProLeu Gln Trp Leu Cys Glu 1 5 10 15 Lys Tyr Phe Gly 28 11 PRT Artificialsequence Sequence is synthesized 28 Cys Arg Ala Gly Pro Leu Gln Trp LeuCys Glu 1 5 10 29 14 PRT Artificial sequence Sequence is synthesized 29Cys Arg Lys Gly Pro Leu Gln Trp Leu Cys Glu Leu Tyr Phe 1 5 10 30 14 PRTArtificial sequence Sequence is synthesized 30 Cys Arg Lys Gly Pro LeuGln Trp Leu Cys Glu Lys Tyr Phe 1 5 10 31 14 PRT Artificial sequenceSequence is synthesized 31 Cys Lys Glu Gly Pro Leu Gln Trp Leu Cys GluLys Tyr Phe 1 5 10 32 14 PRT Artificial sequence Sequence is synthesized32 Cys Lys Glu Gly Pro Leu Leu Trp Leu Cys Glu Lys Tyr Phe 1 5 10 33 19PRT Artificial sequence Sequence is synthesized 33 Ser Glu Val Gly CysArg Ala Gly Pro Leu Gln Trp Leu Cys Glu 1 5 10 15 Lys Tyr Phe Gly 34 14PRT Artificial sequence Sequence is synthesized 34 Cys Ala Ala Gly ProLeu Gln Trp Leu Cys Glu Lys Tyr Phe 1 5 10 35 14 PRT Artificial sequenceSequence is synthesized 35 Cys Arg Ala Gly Pro Leu Gln Trp Leu Cys GluLys Tyr Phe 1 5 10 36 12 PRT Artificial sequence Sequence is synthesized36 Cys Arg Ala Gly Pro Leu Gln Trp Leu Cys Glu Lys 1 5 10 37 14 PRTArtificial sequence Sequence is synthesized 37 Cys Arg Ala Gly Pro LeuGln Trp Leu Cys Glu Lys Ala Ala 1 5 10 38 18 PRT Artificial sequenceSequence is synthesized 38 Ser Glu Met Val Cys Arg Ala Gly Pro Leu GlnTrp Leu Cys Glu 1 5 10 15 Ile Tyr Phe 39 18 PRT Artificial sequenceSequence is synthesized 39 Glu Ala Arg Val Cys Arg Ala Gly Pro Leu GlnTrp Leu Cys Glu 1 5 10 15 Lys Tyr Phe 40 21 PRT Artificial sequenceSequence is synthesized 40 Ser Glu Val Gly Cys Arg Ala Gly Pro Leu GlnTrp Leu Cys Glu 1 5 10 15 Lys Tyr Phe Ser Thr Tyr 20 41 17 PRTArtificial sequence Sequence is synthesized 41 Cys Arg Ala Gly Pro LeuGln Trp Leu Cys Glu Lys Tyr Phe Ser 1 5 10 15 Thr Tyr 42 21 PRTArtificial sequence Sequence is synthesized 42 Glu Ala Arg Val Cys ArgAla Gly Pro Leu Gln Trp Leu Cys Glu 1 5 10 15 Lys Tyr Phe Ser Thr Tyr 2043 21 PRT Artificial sequence Sequence is synthesized 43 Gly Gln Gln SerCys Arg Ala Gly Pro Leu Gln Trp Leu Cys Glu 1 5 10 15 Lys Tyr Phe SerThr Tyr 20 44 14 PRT Artificial sequence Sequence is synthesized 44 CysArg Ala Gly Pro Leu Gln Trp Leu Cys Glu Arg Tyr Phe 1 5 10 45 14 PRTArtificial sequence Sequence is synthesized 45 Cys Arg Ala Gly Pro LeuGln Trp Leu Cys Glu Lys Phe Phe 1 5 10 46 23 PRT Artificial sequenceSequence is synthesized 46 Gly Gln Gln Ser Cys Ala Ala Gly Pro Leu GlnTrp Leu Cys Glu 1 5 10 15 His Tyr Phe Ser Thr Tyr Gly Arg 20 47 97 PRTArtificial sequence Sequence is synthesized 47 Gly Gln Gln Ser Cys AlaAla Gly Pro Leu Gln Trp Leu Cys Glu 1 5 10 15 His Tyr Phe Ser Thr TyrGly Arg Gly Gly Gly Ser Gly Gly Ala 20 25 30 Gln His Asp Glu Ala Val AspAsn Lys Phe Asn Lys Glu Gln Gln 35 40 45 Asn Ala Phe Tyr Glu Ile Leu HisLeu Pro Asn Leu Asn Glu Glu 50 55 60 Gln Arg Asn Ala Phe Ile Gln Ser LeuLys Asp Asp Pro Ser Gln 65 70 75 Ser Ala Asn Leu Leu Ala Glu Ala Lys LysLeu Asn Asp Ala Gln 80 85 90 Ala Pro Asn Val Asp Met Asn 95 48 14 PRTArtificial sequence Sequence is synthesized 48 Cys Lys Ala Gly Pro LeuLeu Trp Leu Cys Glu Arg Phe Phe 1 5 10 49 14 PRT Artificial sequenceSequence is synthesized 49 Cys Arg Ala Gly Pro Leu Gln Trp Leu Cys GluArg Phe Phe 1 5 10 50 14 PRT Artificial sequence Sequence is synthesized50 Cys Arg Glu Gly Pro Leu Gln Trp Leu Cys Glu Arg Phe Phe 1 5 10 51 14PRT Artificial sequence Sequence is synthesized 51 Cys Lys Glu Gly ProLeu Leu Trp Leu Cys Glu Arg Phe Phe 1 5 10 52 14 PRT Artificial sequenceSequence is synthesized 52 Arg Ala Gly Pro Leu Glu Trp Leu Ala Glu LysTyr Glu Gly 1 5 10 53 12 PRT Artificial sequence Sequence is synthesized53 Arg Pro Leu Glu Trp Leu Ala Glu Lys Tyr Phe Glu 1 5 10 54 14 PRTArtificial sequence Sequence is synthesized 54 Arg Ala Gly Pro Leu GluTrp Leu Ala Glu Lys Tyr Phe Glu 1 5 10 55 77 PRT Artificial sequenceSequence is synthesized 55 Ser Gly Thr Ala Met Ala Asp Pro Asn Arg PheArg Gly Lys Asp 1 5 10 15 Leu Ala Gly Ser Pro Gly Gly Gly Ser Gly GlyGly Ala Glu Gly 20 25 30 Asp Asp Pro Ala Lys Ala Ala Phe Asn Ser Leu GlnAla Ser Ala 35 40 45 Thr Glu Tyr Ile Gly Tyr Ala Trp Ala Met Val Val ValIle Val 50 55 60 Gly Ala Thr Ile Gly Ile Lys Leu Phe Lys Lys Phe Thr SerLys 65 70 75 Ala Ser 56 16 PRT Artificial sequence Sequence issynthesized 56 Ser Gly Thr Ala Cys Xaa Gly Pro Xaa Cys Ser Leu Ala GlySer 1 5 10 15 Pro 57 18 PRT Artificial sequence Sequence is synthesized57 Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15Xaa Xaa Xaa 58 20 PRT Artificial sequence Sequence is synthesized 58 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa 20 59 20 PRT Artificial sequence Sequence is synthesized59 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Xaa 20 60 20 PRT Artificial sequence Sequence issynthesized 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa CysXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 61 20 PRT Artificial sequenceSequence is synthesized 61 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 62 20 PRT Artificialsequence Sequence is synthesized 62 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 63 20 PRTArtificial sequence Sequence is synthesized 63 Xaa Xaa Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 6420 PRT Artificial sequence Sequence is synthesized 64 Xaa Xaa Xaa XaaXaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa XaaXaa 20 65 20 PRT Artificial sequence Sequence is synthesized 65 Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Cys XaaXaa Xaa Xaa 20 66 20 PRT Artificial sequence Sequence is synthesized 66Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys 1 5 10 15Lys Pro Gln Gly Gly 20 67 10 PRT Artificial sequence Sequence issynthesized 67 Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys 1 5 10 68 70 PRTArtificial sequence Sequence is synthesized 68 Gly Cys Cys Thr Ala ThrGly Cys Ala Thr Cys Thr Gly Gly Thr 1 5 10 15 Ala Cys Cys Gly Cys CysThr Gly Cys Asn Asn Ser Asn Asn Ser 20 25 30 Gly Gly Thr Cys Cys Thr AsnAsn Ser Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Thr Gly Thr Thr CysThr Cys Thr Gly Gly Cys Ala 50 55 60 Gly Gly Thr Thr Cys Ala Cys Cys AlaGly 65 70 69 91 PRT Artificial sequence Sequence is synthesized 69 GlyCys Thr Ala Cys Ala Ala Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 GlyCys Ala Asn Asn Ser Asn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Thr GlyCys Asn Asn Ser Asn Asn Ser Gly Gly Thr Cys Cys Thr 35 40 45 Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser Thr Gly Thr 50 55 60 Asn Asn Ser AsnAsn Ser Asn Asn Ser Asn Asn Ser Gly Gly Thr 65 70 75 Gly Gly Ala Gly GlyAla Thr Cys Cys Gly Gly Ala Gly Gly Ala 80 85 90 Gly 70 97 PRTArtificial sequence Sequence is synthesized 70 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Asn Asn Ser AsnAsn Ser Thr Gly Cys Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Thr Gly Cys Asn Asn Ser 50 55 60 Asn Asn Ser Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 71 97 PRTArtificial sequence Sequence is synthesized 71 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Asn Asn Ser AsnAsn Ser Thr Gly Cys Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Thr Gly Cys 50 55 60 Asn Asn Ser Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 72 97 PRTArtificial sequence Sequence is synthesized 72 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Asn Asn Ser ThrGly Cys Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Thr Gly Cys 50 55 60 Asn Asn Ser Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 73 97 PRTArtificial sequence Sequence is synthesized 73 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Asn Asn Ser ThrGly Cys Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Asn Asn Ser 50 55 60 Thr Gly Cys Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 74 97 PRTArtificial sequence Sequence is synthesized 74 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Thr Gly Cys AsnAsn Ser Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Asn Asn Ser 50 55 60 Thr Gly Cys Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 75 97 PRTArtificial sequence Sequence is synthesized 75 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Thr Gly Cys AsnAsn Ser Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Asn Asn Ser 50 55 60 Asn Asn Ser Thr Gly Cys Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 76 97 PRTArtificial sequence Sequence is synthesized 76 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Thr Gly Cys Asn Asn Ser AsnAsn Ser Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Asn Asn Ser 50 55 60 Asn Asn Ser Thr Gly Cys Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 77 97 PRTArtificial sequence Sequence is synthesized 77 Gly Cys Thr Ala Cys AlaAla Ala Thr Gly Cys Cys Thr Ala Thr 1 5 10 15 Gly Cys Ala Asn Asn SerAsn Asn Ser Asn Asn Ser Asn Asn Ser 20 25 30 Asn Asn Ser Asn Asn Ser AsnAsn Ser Asn Asn Ser Asn Asn Ser 35 40 45 Asn Asn Ser Asn Asn Ser Asn AsnSer Asn Asn Ser Asn Asn Ser 50 55 60 Asn Asn Ser Asn Asn Ser Asn Asn SerAsn Asn Ser Asn Asn Ser 65 70 75 Asn Asn Ser Gly Gly Thr Gly Gly Ala GlyGly Ala Thr Cys Cys 80 85 90 Gly Gly Ala Gly Gly Ala Gly 95 78 20 PRTArtificial sequence Sequence is synthesized 78 Ser Gly Thr Ala Cys TyrGly Gly Pro Glu Trp Trp Cys Cys Ser 1 5 10 15 Leu Ala Gly Ser Pro 20 7920 PRT Artificial sequence Sequence is synthesized 79 Ser Gly Thr AlaCys Tyr Gly Gly Pro Glu Trp Trp Cys Cys Ser 1 5 10 15 Leu Ala Gly SerPro 20 80 20 PRT Artificial sequence Sequence is synthesized 80 Ser GlyThr Ala Cys Tyr Gly Gly Pro Glu Trp Trp Cys Cys Ser 1 5 10 15 Leu AlaGly Ser Pro 20 81 18 PRT Artificial sequence Sequence is synthesized 81Asp Leu Ala Ile Cys Ala Glu Gly Pro Glu Ile Trp Val Cys Glu 1 5 10 15Glu Thr Ser 82 18 PRT Artificial sequence Sequence is synthesized 82 AspPhe Trp Ile Cys Leu Ser Gly Pro Gly Trp Glu Glu Cys Leu 1 5 10 15 GluTrp Trp 83 18 PRT Artificial sequence Sequence is synthesized 83 Glu GluSer Glu Cys Phe Glu Gly Pro Gly Tyr Val Ile Cys Gly 1 5 10 15 Leu ValGly 84 18 PRT Artificial sequence Sequence is synthesized 84 Asp Met GlyVal Cys Ala Asp Gly Pro Trp Met Tyr Val Cys Glu 1 5 10 15 Trp Thr Glu 8518 PRT Artificial sequence Sequence is synthesized 85 Asp Met Gly ValCys Ala Asp Gly Pro Trp Met Tyr Val Cys Glu 1 5 10 15 Trp Thr Glu 86 20PRT Artificial sequence Sequence is synthesized 86 Gly Ser Ala Gly GlnGly Met Thr Glu Glu Trp Ala Trp Ile Trp 1 5 10 15 Glu Trp Trp Lys Glu 2087 20 PRT Artificial sequence Sequence is synthesized 87 Glu Leu Asp GlyTrp Val Cys Ile Lys Val Gly Glu Gln Asn Leu 1 5 10 15 Cys Tyr Leu AlaGlu 20 88 20 PRT Artificial sequence Sequence is synthesized 88 Glu LeuAsp Gly Trp Val Cys Ile Lys Val Gly Glu Gln Asn Leu 1 5 10 15 Cys TyrLeu Ala Glu 20 89 20 PRT Artificial sequence Sequence is synthesized 89Ala Ile Gly Gly Trp Cys Phe Ile Glu Leu Asp Ser Leu Trp Cys 1 5 10 15Glu Glu Gln Ile Gly 20 90 20 PRT Artificial sequence Sequence issynthesized 90 Ser Glu Asp Val Glu Cys Trp Gln Val Trp Glu Asn Leu ValCys 1 5 10 15 Ser Val Glu His Arg 20 91 19 PRT Artificial sequenceSequence is synthesized 91 Ser Glu Glu Val Cys Trp Pro Val Ala Glu TrpTyr Leu Cys Asn 1 5 10 15 Met Trp Gly Arg 92 20 PRT Artificial sequenceSequence is synthesized 92 Arg Val Gly Ala Tyr Ile Ser Cys Ser Glu ThrGlu Cys Trp Val 1 5 10 15 Glu Asp Leu Leu Asp 20 93 20 PRT Artificialsequence Sequence is synthesized 93 Trp Phe Lys Thr Val Cys Tyr Glu TrpGlu Asp Glu Val Gln Cys 1 5 10 15 Tyr Thr Leu Glu Glu 20 94 20 PRTArtificial sequence Sequence is synthesized 94 Ser Glu Asp Val Glu CysTrp Gln Val Trp Glu Asn Leu Val Cys 1 5 10 15 Ser Val Glu His Arg 20 9520 PRT Artificial sequence Sequence is synthesized 95 Arg Leu Glu GluGln Cys Val Glu Val Asn Tyr Glu Pro Ser Cys 1 5 10 15 Ser Phe Thr AlaAsn 20 96 19 PRT Artificial sequence Sequence is synthesized 96 Ser GluGlu Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 15 Ile LeuGly Pro 97 20 PRT Artificial sequence Sequence is synthesized 97 Glu ThrVal Ala Asn Cys Asp Cys Tyr Met Asp Leu Cys Leu Cys 1 5 10 15 Tyr GlySer Asp Arg 20 98 20 PRT Artificial sequence Sequence is synthesized 98Tyr His Pro Ile Ser Cys Met Asp His Tyr Tyr Leu Ile Ile Cys 1 5 10 15Asp Glu Thr Val Asn 20 99 20 PRT Artificial sequence Sequence issynthesized 99 Val Ala Trp Glu Val Cys Trp Asp Arg His Asp Gln Gly TyrIle 1 5 10 15 Cys Thr Thr Asp Ser 20 100 20 PRT Artificial sequenceSequence is synthesized 100 Ala Glu Trp Ala Glu Cys Trp Ile Ala Gly AspGln Leu Leu Cys 1 5 10 15 Val Gly Lys Asp Asn 20 101 20 PRT Artificialsequence Sequence is synthesized 101 Glu Pro Trp Leu Cys Gln Tyr Tyr GluAla Ala Met Leu Tyr Leu 1 5 10 15 Cys Trp Glu Glu Gly 20 102 20 PRTArtificial sequence Sequence is synthesized 102 Ala Glu Glu Gly Met ValTrp Gly Trp Thr Gly Gly Trp Tyr Asn 1 5 10 15 Leu Asp Glu Leu Cys 20 10320 PRT Artificial sequence Sequence is synthesized 103 Ser Gly Gly AlaIle Tyr Trp Pro Val Glu Gln Phe Ile Ala Phe 1 5 10 15 Met Ala Val GlyLys 20 104 20 PRT Artificial sequence Sequence is synthesized 104 GluPro Trp Leu Cys Gln Tyr Tyr Glu Ala Ala Met Leu Tyr Leu 1 5 10 15 CysTrp Glu Glu Gly 20 105 20 PRT Artificial sequence Sequence issynthesized 105 Ser Gly Gly Ala Ile Tyr Met Pro Val Glu Gln Phe Ile AlaPhe 1 5 10 15 Met Ala Val Gly Lys 20 106 18 PRT Artificial sequenceSequence is synthesized 106 Glu Val Leu Leu Cys Ser Asp Gly Pro Gln LeuTyr Leu Cys Glu 1 5 10 15 Leu Tyr Ala 107 18 PRT Artificial sequenceSequence is synthesized 107 Ser Gly Val Glu Cys Val Trp Gly Pro Gln TrpGly Phe Cys Val 1 5 10 15 Glu Glu Tyr 108 18 PRT Artificial sequenceSequence is synthesized 108 Asp Lys Glu Val Cys Tyr Leu Gly Pro Glu ThrTrp Leu Cys Phe 1 5 10 15 Trp Trp Pro 109 18 PRT Artificial sequenceSequence is synthesized 109 Glu Val Leu Leu Cys Ser Asp Gly Pro Gln LeuTyr Leu Cys Glu 1 5 10 15 Leu Tyr Ala 110 18 PRT Artificial sequenceSequence is synthesized 110 Gly Asp Val Glu Cys Ile Glu Gly Pro Trp GlyGlu Leu Cys Val 1 5 10 15 Trp Ala Asp 111 20 PRT Artificial sequenceSequence is synthesized 111 Phe Gly Gly Trp Ser Cys Gln Pro Thr Trp ValAsp Val Tyr Val 1 5 10 15 Cys Asn Phe Glu Glu 20 112 20 PRT Artificialsequence Sequence is synthesized 112 Ala Met Trp Val Cys Val Ser Asp TrpGlu Thr Val Glu Glu Cys 1 5 10 15 Ile Gln Tyr Met Tyr 20 113 20 PRTArtificial sequence Sequence is synthesized 113 Ala Met Trp Val Cys ValSer Asp Trp Glu Thr Val Glu Glu Cys 1 5 10 15 Ile Gln Tyr Met Tyr 20 11420 PRT Artificial sequence Sequence is synthesized 114 Ala Met Trp ValCys Val Ser Asp Trp Glu Thr Val Glu Glu Cys 1 5 10 15 Ile Gln Tyr MetTyr 20 115 20 PRT Artificial sequence Sequence is synthesized 115 AlaMet Trp Val Cys Val Ser Asp Trp Glu Thr Val Glu Glu Cys 1 5 10 15 IleGln Tyr Met Tyr 20 116 20 PRT Artificial sequence Sequence issynthesized 116 Thr Asn Trp Phe Phe Val Cys Glu Ser Gly His Gln Asp IleCys 1 5 10 15 Trp Leu Ala Glu Glu 20 117 18 PRT Artificial sequenceSequence is synthesized 117 Trp Val Met Glu Cys Gly Ala Gly Pro Trp ProGlu Gly Cys Thr 1 5 10 15 Phe Met Leu 118 19 PRT Artificial sequenceSequence is synthesized 118 Arg Lys Thr Ser Gln Gly Arg Gly Gln Glu MetCys Trp Glu Thr 1 5 10 15 Gly Gly Cys Ser 119 20 PRT Artificial sequenceSequence is synthesized 119 Ser Trp Glu Arg Gly Glu Leu Thr Tyr Met LysLeu Cys Glu Tyr 1 5 10 15 Met Arg Leu Gln Gln 20 120 20 PRT Artificialsequence Sequence is synthesized 120 Glu His Gly Arg Ala Asn Cys Leu IleThr Pro Glu Ala Gly Lys 1 5 10 15 Leu Ala Arg Val Thr 20 121 18 PRTArtificial sequence Sequence is synthesized 121 Val Glu Asp Glu Cys TrpMet Gly Pro Asp Trp Ala Val Cys Trp 1 5 10 15 Thr Trp Gly 122 21 PRTArtificial sequence Sequence is synthesized 122 Glu Leu Asp Gly Trp ValCys Ile Lys Val Gly Glu Gln Asn Leu 1 5 10 15 Cys Tyr Leu Ala Glu Gly 20123 21 PRT Artificial sequence Sequence is synthesized 123 Trp Phe LysThr Val Cys Tyr Glu Trp Glu Asp Glu Val Gln Cys 1 5 10 15 Tyr Thr LeuGlu Glu Gly 20 124 21 PRT Artificial sequence Sequence is synthesized124 Arg Val Gly Ala Tyr Ile Ser Cys Ser Glu Thr Glu Cys Trp Val 1 5 1015 Glu Asp Leu Leu Asp Gly 20 125 15 PRT Artificial sequence Sequence issynthesized 125 Cys Trp Asp Arg His Asp Gln Gly Tyr Ile Cys Thr Thr AspSer 1 5 10 15 126 10 PRT Artificial sequence Sequence is synthesized 126Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn 1 5 10 127 38 DNA Artificialsequence Sequence is synthesized 127 agctgctttg atatgcatct cccgaaactctgtgcggt 38 128 37 DNA Artificial sequence Sequence is synthesized 128gagcgatctg ggtctagaca gatttagcgg gtttcag 37 129 24 DNA Artificialsequence Sequence is synthesized 129 aaaagggtat gtagaggttg aggt 24 13014 PRT Artificial sequence Sequence is synthesized 130 Cys Xaa Xaa GlyXaa Leu Xaa Trp Leu Cys Xaa Xaa Xaa Xaa 1 5 10 131 14 PRT Artificialsequence Sequence is synthsized 131 Cys Arg Ala Ala Pro Leu Gln Trp LeuCys Glu Lys Tyr Phe 1 5 10 132 14 PRT Artificial sequence Sequence issynthesized 132 Cys Arg Ala Gly Ala Leu Gln Trp Leu Cys Glu Lys Tyr Phe1 5 10 133 14 PRT Artificial sequence Sequence is synthesized 133 CysArg Ala Gly Arg Leu Gln Trp Leu Cys Glu Lys Tyr Phe 1 5 10 134 14 PRTArtificial sequence Sequence is synthesized 134 Cys Arg Ala Gly Asn LeuGln Trp Leu Cys Glu Lys Tyr Phe 1 5 10 135 14 PRT Artificial sequenceSequence is synthesized 135 Cys Arg Ala Gly Pro Arg Gln Trp Leu Cys GluLys Tyr Phe 1 5 10 136 14 PRT Artificial sequence Sequence issynthesized 136 Cys Arg Ala Gly Pro Xaa Gln Trp Leu Cys Glu Lys Tyr Phe1 5 10 137 14 PRT Artificial sequence Sequence is synthesized 137 CysArg Ala Gly Pro Leu Gln Trp Arg Cys Glu Lys Tyr Phe 1 5 10 138 14 PRTArtificial sequence Sequence is synthesized 138 Cys Arg Ala Gly Pro LeuGln Trp Xaa Cys Glu Lys Tyr Phe 1 5 10 139 14 PRT Artificial sequenceSequence is synthesized 139 Cys Arg Ala Gly Pro Leu Gln Arg Leu Cys GluLys Tyr Phe 1 5 10 140 14 PRT Artificial sequence Sequence issynthesized 140 Cys Arg Ala Gly Pro Leu Gln Xaa Leu Cys Glu Lys Tyr Phe1 5 10 141 14 PRT Artificial sequence Sequence is synthesized 141 CysArg Ala Gly Pro Leu Gln His Leu Cys Glu Lys Tyr Phe 1 5 10 142 19 PRTArtificial sequence Sequence is synthesized 142 Xaa Xaa Xaa Xaa Xaa XaaXaa Pro Leu Glu Xaa Leu Ala Xaa Xaa 1 5 10 15 Xaa Xaa Glu Xaa 143 15 PRTArtificial sequence Sequence is synthesized 143 Cys Arg Ala Gly Pro LeuGln Glu Leu Cys Glu Lys Tyr Ala Glu 1 5 10 15 144 10 PRT Artificialsequence Sequence is synthesized 144 Leu Glu Trp Leu Ala Glu Lys Tyr GluGly 1 5 10 145 11 PRT Artificial sequence Sequence is synthesized 145Pro Leu Glu Trp Leu Ala Glu Lys Tyr Glu Gly 1 5 10 146 10 PRT Artificialsequence Sequence is synthesized 146 Leu Glu Trp Leu Ala Glu Lys Tyr PheGlu 1 5 10 147 4 PRT Artificial sequence Sequence is synthesized 147 GlyGln Gln Ser 1 148 4 PRT Artificial sequence Sequence is synthesized 148Ser Glu Val Gly 1 149 4 PRT Artificial sequence Sequence is synthesized149 Ser Glu Met Val 1 150 4 PRT Artificial sequence Sequence issynthesized 150 Glu Ala Arg Val 1 151 4 PRT Artificial sequence Sequenceis synthesized 151 Gly Gln Gln Ser 1 152 18 PRT Artificial sequenceSequence is synthesized 152 Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Xaa Leu XaaXaa Leu Cys Xaa 1 5 10 15 Xaa Xaa Xaa 153 21 PRT Artificial sequenceSequence is synthesized 153 Gly Gln Gln Ser Cys Ala Ala Gly Pro Leu GlnTrp Leu Cys Glu 1 5 10 15 His Tyr Phe Ser Thr Tyr 20

What is claimed is:
 1. A method of treating cartilage disorderscomprising contacting cartilage with an effective amount of an activeagent selected from an IGF-1 analog with a binding affinity preferencefor insulin-like growth factor binding protein-3 (IGFBP-3) overinsulin-like growth factor binding protein-1 (IGFBP-1), an IGF-1 analogwith a binding affinity preference for IGFBP-1 over IGFBP-3, or a IGFBPdisplacer peptide that prevents the interaction of IGF with an IGFBP anddoes not bind to a human IGF receptor.
 2. The method of claim 1 whereinthe disorder is found in a mammal and the active agent is administeredto the mammal.
 3. The method of claim 2 wherein the active agent isadministered locally to the cartilage.
 4. The method of claim 3 whereinthe active agent is an IGF-1 analog with a binding affinity preferencefor IGFBP-3 over IGFBP-1 or an IGFBP-3 displacer peptide, which furthercomprises administering to the mammal an effective amount of IGF-1,acid-labile subunit (ALS), or IGF-1 and ALS.
 5. The method of claim 4wherein the active agent and IGF-1 or ALS, or the active agent, IGF-1,and ALS are administered together as a complex.
 6. The method of claim 2wherein the mammal is human.
 7. The method of claim 1 wherein the analogis an IGF-1 variant wherein the amino acid residue at position 3, 7, 10,16, 25, or 49 or the amino acid residues at positions 3 and 49 ofnative-sequence human IGF-1 are replaced with an alanine, a glycine, ora serine residue.
 8. The method of claim 1 wherein the analog is anIGF-1 variant wherein the amino acid residue at position 9, 12, 15, or20 of native-sequence human IGF-1 is replaced with a lysine or arginineresidue.
 9. The method of claim 1 wherein the peptide is an IGFBP-3 orIGFBP-1 displacer peptide.
 10. The method of claim 9 wherein the peptideis an IGFBP-3 displacer peptide selected from the group consisting of:Y24LY31A; 4D3.3P (SEQ ID NO:6); BP3-4D3.11 (SEQ ID NO:7); BP3-4D3.11DEL(SEQ ID NO:8); BP3-4B3.3 (SEQ ID NO:9); BP3-01-ox (SEQ ID NO:10);BP3-02-ox (SEQ ID NO:11); BP3-06 (SEQ ID NO:12); BP3-08 (SEQ ID NO:13);BP3-15 (SEQ ID NO:14); BP3-16 (SEQ ID NO:15); BP3-17 (SEQ ID NO:16);BP3-25 (SEQ ID NO:17); BP3-27 (SEQ ID NO:18); BP3-28 (SEQ ID NO:19);BP3-30 (SEQ ID NO:20); BP3-39 (SEQ ID NO:21); BP3-40 (SEQ ID NO:22);BP3-41 (SEQ ID NO:23); BP3-107 (SEQ ID NO:24); and BP3-108 (SEQ IDNO:25).
 11. The method of claim 10 wherein the peptide is BP3-15,BP3-39, BP3-40, BP3-01-OX, BP3-27, BP3-28, BP3-30, BP3-41, or 4D3.3P.12. The method of claim 11 wherein the peptide is BP3-15, BP3-39, orBP3-40.
 13. The method of claim 9 wherein the peptide is an IGFBP-1displacer peptide selected from the group consisting of: BP1-01 (SEQ IDNO:26); BP1-02 (SEQ ID NO:27); BP1-04 (SEQ ID NO:28); BP1-10 (SEQ IDNO:29); BP1-11 (SEQ ID NO:30); BP1-12 (SEQ ID NO:31); BP1-13 (SEQ IDNO:32); BP1-14 (SEQ ID NO:33); BP1-15 (SEQ ID NO:34); BP1-16 (SEQ IDNO:35); BP1-17 (SEQ ID NO:36); BP1-18 (SEQ ID NO:37); BP1-19 (SEQ IDNO:38); BP1-20 (SEQ ID NO:39); BP1-21A (SEQ ID NO:40); BP1-21B (SEQ IDNO:41); BP1-25 (SEQ ID NO:42); BP1-40 (SEQ ID NO:43); BP67 (SEQ IDNO:44); BP68 (SEQ ID NO:45); BP1-625 (SEQ ID NO:46); BP1-625-Z (SEQ IDNO:47); BP1-625T (SEQ ID NO:153); BP1027 (SEQ ID NO:48); BP1028 (SEQ IDNO:49); BP1029 (SEQ ID NO:50); BP1030 (SEQ ID NO:51); (i+7)D (SEQ IDNO:52); (i+8)B (SEQ ID NO:53); and (i+8)C (SEQ ID NO:54).
 14. The methodof claim 13 wherein the peptide is BP1-16, BP1-20, BP1-21A, BP1-25,BP1-40, BP625, BP625-Z, or BP625T.
 15. The method of claim 14 whereinthe peptide is BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, BP1-625-Z, orBP1-625T.
 16. The method of claim 1 wherein the active agent is F49A,E3A, F16A, E3AF49A, F16AF49A, D12K, D12R, BP3-15, BP3-40, BP3-39,BP1-16, BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, or BP1-625-Zj.
 17. Themethod of claim 1 wherein the active agent is F49A, E3AF49A, F16AF49A,D12K, D12R, BP3-15, BP3-40, BP3-39, BP1-20, BP1-21A, BP1-25, BP1-40,BP1-625, BP1-625-Z, or BP1-625T.
 18. The method of claim 1 wherein thedisorder is a degenerative cartilagenous disorder.
 19. The method ofclaim 18 wherein the disorder is an articular cartilage disorder. 20.The method of claim 19 wherein the articular cartilage disorder isselected from the group consisting of rheumatoid arthritis andosteoarthritis.
 21. The method of claim 1, wherein the active agent isadministered by direct injection into the afflicted cartilagenous regionor joint.
 22. The method of claim 1, wherein the active agent is in anextended-release formulation.
 23. The method of claim 1, furthercomprising contacting the cartilage with an effective amount of acartilage growth factor or a cartilage catabolism antagonist.
 24. Themethod of claim 23 wherein the cartilage growth factor is IGF-1.
 25. Anarticle of manufacture comprising a container holding an active agentselected from an IGF-1 analog with a binding affinity preference forIGFBP-3 over IGFBP-1, an IGF-1 analog with a binding affinity preferencefor IGFBP-1 over IGFBP-3, or an IGFBP displacer peptide that preventsthe interaction of IGF with an IGFBP and does not bind to a human IGFreceptor in a pharmaceutically acceptable carrier with instructions forits use in treating a cartilage disorder.
 26. The article of claim 25further comprising a container holding a cartilage growth factor or acartilage catabolism antagonist.